Hcv protease inhibitors and uses thereof

ABSTRACT

The present invention provides compounds, pharmaceutically acceptable compositions thereof, and methods of using the same.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation-in-part of U.S. Ser. No.12/339,770, filed Dec. 19, 2008, which claims priority to U.S.provisional application Ser. No. 61/016,110, filed Dec. 21, 2007, U.S.provisional application Ser. No. 61/016,473, filed Dec. 23, 2007, U.S.provisional application Ser. No. 61/075,001, filed Jun. 23, 2008, andU.S. provisional application Ser. No. 61/098,675, filed Sep. 19, 2008,the entirety of each of which is hereby incorporated by reference.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to compounds useful as inhibitors of HCVprotease. The invention also provides pharmaceutically acceptablecompositions comprising compounds of the present invention and methodsof using said compositions in the treatment of various disorders.

BACKGROUND OF THE INVENTION

It is estimated that over 170 million people worldwide are infected withthe Hepatitis C virus (HCV). With an estimated human sero-prevalence of3% globally, HCV is the major cause for most cases of non-A, non-Bhepatitis, (Alberti, A. et al., J. Hepatology 31, (Suppl. 1): 17-24,1999). While the symptoms of acute hepatitis subside in some patients,at least 85% of HCV infections become chronic, and 20% of those infecteddevelop liver cirrhosis. There is less than a 50% survival rate at fouryears post cirrhosis diagnosis. Chronic HCV infection is also associatedwith increased incidence of hepatocellular carcinoma.

HCV is a positive-stranded RNA virus whose genome encodes a polyproteinof approximately 3000 amino acids. This precursor protein is processedinto at least 10 viral structural and nonstructural proteins: C, E1, E2,p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B (Blight, K. J., et al.,Antiviral Ther. 3, Suppl. 3: 71-81, 1998). HCV nonstructural (NS)proteins are derived by proteolytic cleavage of the polyprotein and arepresumed to provide the essential catalytic machinery for viralreplication.

NS3 is an approximately 68 Kda protein, and has both an N-terminalserine protease domain and an RNA-dependent ATPase domain at itsC-terminus. It has been shown that the NS4A protein serves as aco-factor for the serine protease activity of NS3. NS3 functions as aproteolytic enzyme that cleaves sites liberating other nonstructuralproteins necessary for HCV replication and is a viable therapeutictarget for antiviral chemotherapy.

No vaccines are available for HCV, and the established therapy ofinterferon treatment is effective in only 15-20% of patients (Weiland,O., FEMS Microbiol. Rev. 14: 279-88, 1994), and has significant sideeffects (Walker, M. A., et al., DDT 4: 518-29, 1999; Moradpour, D., etal., Eur. J. Gastroenterol. Hepatol. 11: 1199-1202, 1999). While thecurrent standard of care, pegylated interferon α in combination withribavirin, is more efficacious and appears to decrease hepatocellularcarcinoma in patients with HCV-related cirrhosis (Hung, C. H., et al., JViral Hepatitis 13(6): 409-414, 2006), this treatment has also beenshown to produce side effects such as thyroid dysfunction (Huang, J. F.,et al., J Viral Hepatitis 13(6): 396-401, 2006).

The poor prognosis for patients suffering from HCV infection and thecurrent lack of effective, approved treatments, highlights theoverwhelming need for new inhibitors of HCV NS3 protease.

SUMMARY OF THE INVENTION

It has now been found that compounds of this invention, andpharmaceutically acceptable compositions thereof, are effective asinhibitors of HCV protease. Such compounds have the general formula I:

or a pharmaceutically acceptable salt thereof, wherein R¹, R^(1′),R^(2a), R³, R⁴ and R^(z) are as defined herein.

Compounds of the present invention, and pharmaceutically acceptablecompositions thereof, are useful for treating a variety of diseases,disorders or conditions, associated with HCV. Such diseases, disorders,or conditions include those described herein.

Compounds provided by this invention are also useful for the study ofHCV protease in biological and pathological phenomena; the study ofintracellular signal transduction pathways mediated by HCV protease; andthe comparative evaluation of new HCV protease inhibitors.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a mass spectroscopic analysis of HCV NS3/4A wild-typeprotease in the presence of test compound I-1.

FIG. 2 depicts a mass spectroscopic analysis of HCV NS3/4A wild-typeprotease in the presence of test compound I-25.

FIG. 3 depicts a mass spectroscopic analysis of HCV NS3/4A protease.

FIG. 4 depicts a mass spectroscopic analysis of HCV NS3/4A mutant D168Vprotease in the presence of test compound I-11.

FIG. 5 depicts a mass spectroscopic analysis of HCV NS3/4A mutant A156Sprotease in the presence of test compound I-11.

FIG. 6 depicts a mass spectroscopic analysis of HCV NS3/4A mutant R155Kprotease in the presence of test compound I-11.

FIG. 7 depicts a mass spectroscopic analysis of HCV NS3/4A mutant A156Tprotease in the presence of test compound I-11.

FIG. 8 depicts that the NS3 internal self-cleavage products areinhibited by treatment of replicon cells with Compound I-47 for 16hours.

FIGS. 9 and 9-A depict an irreversible covalent inhibitor (compoundI-11) of NS3 protease demonstrates prolonged inhibition of NS3 proteaseactivity in the wild-type replicon cells, as measured by self-cleavage,after the compounds are removed.

FIG. 10 depicts an irreversible covalent inhibitor (compound I-25) ofNS3 protease demonstrate prolonged inhibition of NS3 protease activityin the wild-type replicon cells, as measured by self-cleavage, after thecompounds are removed.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS 1. General Description ofCompounds of the Invention

In certain embodiments, the present invention provides a compound offormula I:

or a pharmaceutically acceptable salt thereof, wherein:

-   R¹ and R^(1′) are independently hydrogen or optionally substituted    C₁₋₆ aliphatic, or R¹ and R^(1′) are taken together to form an    optionally substituted 3-7 membered carbocyclic ring;-   R^(2a) is —OH or —NHSO₂R²;-   R² is —N(R)₂ or an optionally substituted group selected from C₃₋₇    cycloalkyl, a bridged bicyclic, 6-10 membered aryl, 5-10 membered    heteroaryl having 1-4 heteroatoms independently selected from    nitrogen, oxygen, or sulfur, or 4-7 membered heterocyclyl having 1-2    heteroatoms independently selected from nitrogen, oxygen, or sulfur;-   each R is independently hydrogen, optionally substituted C₁₋₆    aliphatic, or:    -   two R on the same nitrogen atom are taken together with the        nitrogen to form a 4-7 membered heterocyclic ring having 1-2        heteroatoms independently selected from nitrogen, oxygen, or        sulfur;-   R³ is a warhead group, or:    -   R³ and R¹ are taken together with their intervening atoms to        form an optionally substituted saturated or unsaturated 12-18        membered ring having 2-6 heteroatoms independently selected from        nitrogen oxygen, or sulfur, wherein the ring formed thereby        comprises a warhead group; or    -   R³ and a ring formed by R¹ and R^(1′) are taken together with        their intervening atoms to form an optionally substituted        saturated or unsaturated 12-18 membered ring having 2-6        heteroatoms independently selected from nitrogen oxygen, or        sulfur, wherein the ring formed thereby comprises a warhead        group;-   R⁴ is H, —NHC(O)R⁵, —NHC(O)OR⁶,

or a natural or unnatural amino acid side-chain group;

-   each R⁵ is independently —N(R)₂ or an optionally substituted group    selected from C₁₋₆ aliphatic, a bridged bicyclic, 6-10 membered    aryl, 5-10 membered heteroaryl having 1-4 heteroatoms independently    selected from nitrogen, oxygen, or sulfur, or 4-7 membered    heterocyclyl having 1-2 heteroatoms independently selected from    nitrogen, oxygen, or sulfur;-   R⁶ is an optionally substituted group selected from C₁₋₆ aliphatic,    a bridged bicyclic, 6-10 membered aryl, 5-10 membered heteroaryl    having 1-4 heteroatoms independently selected from nitrogen, oxygen,    or sulfur, or 4-7 membered heterocyclyl having 1-2 heteroatoms    independently selected from nitrogen, oxygen, or sulfur;-   R⁷ is an optionally substituted group selected from C₁₋₆ aliphatic,    a bridged bicyclic, 6-10 membered aryl, 5-10 membered heteroaryl    having 1-4 heteroatoms independently selected from nitrogen, oxygen,    or sulfur, or 4-7 membered heterocyclyl having 1-2 heteroatoms    independently selected from nitrogen, oxygen, or sulfur; and-   R^(z) is

or R⁴ and R^(z) are taken together with their intervening atoms to forman optionally substituted, saturated or unsaturated 16-22 membered ringhaving 2-6 heteroatoms independently selected from nitrogen, oxygen, orsulfur; each occurrence of R^(y) is independently selected from halogen,—OR^(◯), —CN, —NO₂, —N(R^(◯))₂, or optionally substituted C₁₋₄aliphatic; and

-   m is an integer from 0 to 4, inclusive;-   s is an integer from 0 to 4, inclusive;-   t is an integer from 0 to 4, inclusive;    wherein the sum of s and t is non-zero.

2. Compounds and Definitions

Compounds of this invention include those described generally above, andare further illustrated by the classes, subclasses, and speciesdisclosed herein. As used herein, the following definitions shall applyunless otherwise indicated. For purposes of this invention, the chemicalelements are identified in accordance with the Periodic Table of theElements, CAS version, Handbook of Chemistry and Physics, 75^(th) Ed.Additionally, general principles of organic chemistry are described in“Organic Chemistry”, Thomas Sorrell, University Science Books,Sausalito: 1999, and “March's Advanced Organic Chemistry”, 5^(th) Ed.:Smith, M. B. and March, J., John Wiley & Sons, New York: 2001, theentire contents of which are hereby incorporated by reference.

The term “aliphatic” or “aliphatic group”, as used herein, means astraight-chain (i.e., unbranched) or branched, substituted orunsubstituted hydrocarbon chain that is completely saturated or thatcontains one or more units of unsaturation, or a monocyclic hydrocarbonor bicyclic hydrocarbon that is completely saturated or that containsone or more units of unsaturation, but which is not aromatic (alsoreferred to herein as “carbocycle,” “cycloaliphatic” or “cycloalkyl”),that has a single point of attachment to the rest of the molecule.Unless otherwise specified, aliphatic groups contain 1-6 aliphaticcarbon atoms. In some embodiments, aliphatic groups contain 1-5aliphatic carbon atoms. In other embodiments, aliphatic groups contain1-4 aliphatic carbon atoms. In still other embodiments, aliphatic groupscontain 1-3 aliphatic carbon atoms, and in yet other embodiments,aliphatic groups contain 1-2 aliphatic carbon atoms. In someembodiments, “cycloaliphatic” (or “carbocycle” or “cycloalkyl”) refersto a monocyclic C₃-C₆ hydrocarbon that is completely saturated or thatcontains one or more units of unsaturation, but which is not aromatic,that has a single point of attachment to the rest of the molecule.Suitable aliphatic groups include, but are not limited to, linear orbranched, substituted or unsubstituted alkyl, alkenyl, alkynyl groupsand hybrids thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or(cycloalkyl)alkenyl.

As used herein, the term “bridged bicyclic” refers to any bicyclic ringsystem, i.e. carbocyclic or heterocyclic, saturated or partiallyunsaturated, having at least one bridge. As defined by IUPAC, a “bridge”is an unbranched chain of atoms or an atom or a valence bond connectingtwo bridgeheads, where a “bridgehead” is any skeletal atom of the ringsystem which is bonded to three or more skeletal atoms (excludinghydrogen). In some embodiments, a bridged bicyclic group has 7-12 ringmembers and 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur. Such bridged bicyclic groups are well known in theart and include those groups set forth below where each group isattached to the rest of the molecule at any substitutable carbon ornitrogen atom. Unless otherwise specified, a bridged bicyclic group isoptionally substituted with one or more substituents as set forth foraliphatic groups. Additionally or alternatively, any substitutablenitrogen of a bridged bicyclic group is optionally substituted.Exemplary bridged bicyclics include:

The term “lower alkyl” refers to a C₁₋₄ straight or branched alkylgroup. Exemplary lower alkyl groups are methyl, ethyl, propyl,isopropyl, butyl, isobutyl, and tert-butyl.

The term “lower haloalkyl” refers to a C₁₋₄ straight or branched alkylgroup that is substituted with one or more halogen atoms.

The term “heteroatom” means one or more of oxygen, sulfur, nitrogen,phosphorus, or silicon (including, any oxidized form of nitrogen,sulfur, phosphorus, or silicon; the quaternized form of any basicnitrogen or; a substitutable nitrogen of a heterocyclic ring, forexample N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) orNR^(|) (as in N-substituted pyrrolidinyl)).

The term “unsaturated,” as used herein, means that a moiety has one ormore units of unsaturation.

As used herein, the term “bivalent C₁₋₈ (or C₁₋₆) saturated orunsaturated, straight or branched, hydrocarbon chain”, refers tobivalent alkylene, alkenylene, and alkynylene chains that are straightor branched as defined herein.

The term “alkylene” refers to a bivalent alkyl group. An “alkylenechain” is a polymethylene group, i.e., —(CH₂)_(n)—, wherein n is apositive integer, preferably from 1 to 6, from 1 to 4, from 1 to 3, from1 to 2, or from 2 to 3. A substituted alkylene chain is a polymethylenegroup in which one or more methylene hydrogen atoms are replaced with asubstituent. Suitable substituents include those described below for asubstituted aliphatic group.

The term “alkenylene” refers to a bivalent alkenyl group. A substitutedalkenylene chain is a polymethylene group containing at least one doublebond in which one or more hydrogen atoms are replaced with asubstituent. Suitable substituents include those described below for asubstituted aliphatic group.

As used herein, the term “cyclopropylenyl” refers to a bivalentcyclopropyl group of the following structure:

The term “halogen” means F, Cl, Br, or I.

The term “aryl” used alone or as part of a larger moiety as in“aralkyl,” “aralkoxy,” or “aryloxyalkyl,” refers to monocyclic orbicyclic ring systems having a total of five to fourteen ring members,wherein at least one ring in the system is aromatic and wherein eachring in the system contains 3 to 7 ring members. The term “aryl” may beused interchangeably with the term “aryl ring.”

The term “aryl” used alone or as part of a larger moiety as in“aralkyl,” “aralkoxy,” or “aryloxyalkyl,” refers to monocyclic andbicyclic ring systems having a total of five to 10 ring members, whereinat least one ring in the system is aromatic and wherein each ring in thesystem contains three to seven ring members. The term “aryl” may be usedinterchangeably with the term “aryl ring”. In certain embodiments of thepresent invention, “aryl” refers to an aromatic ring system whichincludes, but not limited to, phenyl, biphenyl, naphthyl, anthracyl andthe like, which may bear one or more substituents. Also included withinthe scope of the term “aryl,” as it is used herein, is a group in whichan aromatic ring is fused to one or more non-aromatic rings, such asindanyl, phthalimidyl, naphthimidyl, phenanthridinyl, ortetrahydronaphthyl, and the like.

The terms “heteroaryl” and “heteroar—,” used alone or as part of alarger moiety, e.g., “heteroaralkyl,” or “heteroaralkoxy,” refer togroups having 5 to 10 ring atoms, preferably 5, 6, or 9 ring atoms;having 6, 10, or 14 π electrons shared in a cyclic array; and having, inaddition to carbon atoms, from one to five heteroatoms. The term“heteroatom” refers to nitrogen, oxygen, or sulfur, and includes anyoxidized form of nitrogen or sulfur, and any quaternized form of a basicnitrogen. Heteroaryl groups include, without limitation, thienyl,furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl,oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl,thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl,purinyl, naphthyridinyl, and pteridinyl. The terms “heteroaryl” and“heteroar—”, as used herein, also include groups in which aheteroaromatic ring is fused to one or more aryl, cycloaliphatic, orheterocyclyl rings, where the radical or point of attachment is on theheteroaromatic ring. Nonlimiting examples include indolyl, isoindolyl,benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl,benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl,quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl,phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl,tetrahydroisoquinolinyl, and pyrido[2,3-b]-1,4-oxazin-3(4H)-one. Aheteroaryl group may be mono- or bicyclic. The term “heteroaryl” may beused interchangeably with the terms “heteroaryl ring,” “heteroarylgroup,” or “heteroaromatic,” any of which terms include rings that areoptionally substituted. The term “heteroaralkyl” refers to an alkylgroup substituted by a heteroaryl, wherein the alkyl and heteroarylportions independently are optionally substituted.

As used herein, the terms “heterocycle,” “heterocyclyl,” “heterocyclicradical,” and “heterocyclic ring” are used interchangeably and refer toa stable 5- to 7-membered monocyclic or 7-10-membered bicyclicheterocyclic moiety that is either saturated or partially unsaturated,and having, in addition to carbon atoms, one or more, preferably one tofour, heteroatoms, as defined above. When used in reference to a ringatom of a heterocycle, the term “nitrogen” includes a substitutednitrogen. As an example, in a saturated or partially unsaturated ringhaving 0-3 heteroatoms selected from oxygen, sulfur or nitrogen, thenitrogen may be N (as in 3,4-dihydro-2H-pyrrolyl), NH (as inpyrrolidinyl), or ⁺NR (as in N-substituted pyrrolidinyl).

A heterocyclic ring can be attached to its pendant group at anyheteroatom or carbon atom that results in a stable structure and any ofthe ring atoms can be optionally substituted. Examples of such saturatedor partially unsaturated heterocyclic radicals include, withoutlimitation, tetrahydrofuranyl, tetrahydrothiophenyl pyrrolidinyl,piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl,decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl,diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and quinuclidinyl. Theterms “heterocycle,” “heterocyclyl,” “heterocyclyl ring,” “heterocyclicgroup,” “heterocyclic moiety,” and “heterocyclic radical,” are usedinterchangeably herein, and also include groups in which a heterocyclylring is fused to one or more aryl, heteroaryl, or cycloaliphatic rings,such as indolinyl, 3H-indolyl, chromanyl, phenanthridinyl, ortetrahydroquinolinyl, where the radical or point of attachment is on theheterocyclyl ring. A heterocyclyl group may be mono- or bicyclic. Theterm “heterocyclylalkyl” refers to an alkyl group substituted by aheterocyclyl, wherein the alkyl and heterocyclyl portions independentlyare optionally substituted.

As used herein, the term “partially unsaturated” refers to a ring moietythat includes at least one double or triple bond. The term “partiallyunsaturated” is intended to encompass rings having multiple sites ofunsaturation, but is not intended to include aryl or heteroarylmoieties, as herein defined.

As used herein, the phrase “natural amino acid side-chain group” refersto the side-chain group of any of the 20 amino acids naturally occurringin proteins. Such natural amino acids include the nonpolar, orhydrophobic amino acids, glycine, alanine, valine, leucine isoleucine,methionine, phenylalanine, tryptophan, and proline. Cysteine issometimes classified as nonpolar or hydrophobic and other times aspolar. Natural amino acids also include polar, or hydrophilic aminoacids, such as tyrosine, serine, threonine, aspartic acid (also known asaspartate, when charged), glutamic acid (also known as glutamate, whencharged), asparagine, and glutamine. Certain polar, or hydrophilic,amino acids have charged side-chains. Such charged amino acids includelysine, arginine, and histidine. One of ordinary skill in the art wouldrecognize that protection of a polar or hydrophilic amino acidside-chain can render that amino acid nonpolar. For example, a suitablyprotected tyrosine hydroxyl group can render that tyroine nonpolar andhydrophobic by virtue of protecting the hydroxyl group.

As used herein, the phrase “unnatural amino acid side-chain group”refers to the side-chain group of amino acids not included in the listof 20 amino acids naturally occurring in proteins, as described above.Such amino acids include the D-isomer of any of the 20 naturallyoccurring amino acids. Unnatural amino acids also include homoserine,ornithine, norleucine, and thyroxine. Other unnatural amino acidsside-chains are well known to one of ordinary skill in the art andinclude unnatural aliphatic side chains. Other unnatural amino acidsinclude modified amino acids, including those that are N-alkylated,cyclized, phosphorylated, acetylated, amidated, azidylated, labelled,and the like. In some embodiments, an unnatural amino acid is aD-isomer. In some embodiments, an unnatural amino acid is a L-isomer.

As described herein, compounds of the invention may contain “optionallysubstituted” moieties. In general, the term “substituted,” whetherpreceded by the term “optionally” or not, means that one or morehydrogens of the designated moiety are replaced with a suitablesubstituent. Unless otherwise indicated, an “optionally substituted”group may have a suitable substituent at each substitutable position ofthe group, and when more than one position in any given structure may besubstituted with more than one substituent selected from a specifiedgroup, the substituent may be either the same or different at everyposition. Combinations of substituents envisioned by this invention arepreferably those that result in the formation of stable or chemicallyfeasible compounds. The term “stable,” as used herein, refers tocompounds that are not substantially altered when subjected toconditions to allow for their production, detection, and, in certainembodiments, their recovery, purification, and use for one or more ofthe purposes disclosed herein.

Suitable monovalent substituents on a substitutable carbon atom of an“optionally substituted” group are independently halogen;—(CH₂)₀₋₄R^(◯); —(CH₂)₀₋₄OR^(◯); —O(CH₂)₀₋₄R^(◯), —O—(CH₂)₀₋₄C(O)OR^(◯);—(CH₂)₀₋₄CH(OR^(◯) ₂; —(CH₂)₀₋₄SR^(◯); —(CH₂)₀₋₄Ph, which may besubstituted with R^(◯); —(CH₂)₀₋₄O(CH₂)₀₋₁Ph which may be substitutedwith R^(◯); —CH═CHPh, which may be substituted with R^(◯);—(CH₂)₀₋₄O(CH₂)₀₋₁-pyridyl which may be substituted with R^(◯); NO₂;—CN; —N₃; —(CH₂)₀₋₄N(R^(◯))₂; —(CH₂)₀₋₄N(R^(◯))C(O)R^(◯);—N(R^(◯))C(S)R^(◯); —(CH₂)₀₋₄N(R^(◯))C(O)NR^(◯) ₂;—N(R^(◯))C(S)N(R^(◯))₂; —(CH₂)₀₋₄N(R^(◯))C(O)R^(◯);—N(R^(◯))N(R^(◯))C(O)R^(◯); —N(R^(◯))N(R^(◯))C(O)N(R^(◯))₂;—N(R^(◯))N(R^(◯))C(O)OR^(◯); —(CH₂)₀₋₄C(O)R^(◯); —C(S)R^(◯);—(CH₂)₀₋₄C(O)OR^(◯); —(CH₂)₀₋₄C(O)SR^(◯); —(CH₂)₀₋₄C(O)OSiR^(◯) ₃;—(CH₂)₀₋₄OC(O)R^(◯); —OC(O)CH₂)₀₋₄SR—, SC(S)SR^(◯); —(CH₂)₀₋₄SC(O)R^(◯);—(CH₂)₀₋₄C(O)NR^(◯) ₂; —C(S)NR^(◯) ₂; —C(S)SR^(◯); —SC(S)SR^(◯),—(CH₂)₀₋₄OC(O)NR^(◯) ₂; —C(O)N(OR^(◯))R^(◯); —C(O)C(O)R^(◯);—C(O)CH₂C(O)R^(◯); —C(NOR^(◯))R^(◯); —(CH₂)₀ ₄SSR^(◯); —(CH₂)₀₄S(O)₂R^(◯); —(CH₂)₀₋₄S(O)₂OR^(◯); —(CH₂)₀₋₄OS(O)₂R^(◯); —S(O)₂NR^(◯) ₂;—(CH₂)₀₋₄S(O)R^(◯); —N(R^(◯))S(O)₂NR^(◯) ₂; —N(R^(◯))S(O)₂R^(◯);—N(OR^(◯))R^(◯); —C(NH)NR^(◯) ₂; —P(O)₂R^(◯); —P(O)R^(◯) ₂; —OP(O)R^(◯)₂; —OP(O)(OR^(◯))₂; SiR^(◯) ₃; —(C₁₋₄ straight orbranched)alkylene)O—N(R^(◯))₂; or —(C₁₋₄ straight orbranched)alkylene)C(O)O—N(R^(◯))₂, wherein each R^(◯)may be substitutedas defined below and is independently hydrogen, C₁₋₆ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, —CH₂-(5-6 membered heteroaryl ring), or a 5-6-memberedsaturated, partially unsaturated, or aryl ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, or,notwithstanding the definition above, two independent occurrences ofR^(◯), taken together with their intervening atom(s), form a3-12-membered saturated, partially unsaturated, or aryl mono- orbicyclic ring having 0-4 heteroatoms independently selected fromnitrogen, oxygen, or sulfur, which may be substituted as defined below.

Suitable monovalent substituents on R^(◯)(or the ring formed by takingtwo independent occurrences of R^(◯)together with their interveningatoms), are independently halogen, —(CH₂)₀₋₂R^(), -(haloR^()),—(CH₂)₀₋₂OH, —(CH₂)₀₋₂OR^(), —(CH₂)₀₋₂CH(OR^())₂; —O(haloR^()), —CN,—N₃, —(CH₂)₀₋₂C(O)R^(), —(CH₂)₀₋₂C(O)OH, —(CH₂)₀₋₂C(O)OR^(),—(CH₂)₀₋₂SR^(), —(CH₂)₀₋₂SH, —(CH₂)₀₋₂NH₂, —(CH₂)₀₋₂NHR^(),—(CH₂)₀₋₂NR^() ₂, —NO₂, —SiR^() ₃, —OSiR^() ₃, —C(O)SR^(), —(C₁₋₄straight or branched alkylene)C(O)OR^(), or —SSR^() wherein each R^()is unsubstituted or where preceded by “halo” is substituted only withone or more halogens, and is independently selected from C₁₋₄ aliphatic,—CH₂Ph, —O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partiallyunsaturated, or aryl ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur. Suitable divalent substituents on asaturated carbon atom of R^(◯)include ═O and ═S.

Suitable divalent substituents on a saturated carbon atom of an“optionally substituted” group include the following: ═O, ═S, ═NNR^(*)₂, ═NNHC(O)R^(*), ═NNHC(O)OR^(*), ═NNHS(O)₂R^(*), ═NR^(*), ═NOR,—O(C(R^(*) ₂))₂₋₃O—, or —S(C(R^(*) ₂))₂₋₃S—, wherein each independentoccurrence of R^(*) is selected from hydrogen, C₁₋₆ aliphatic which maybe substituted as defined below, or an unsubstituted 5-6-memberedsaturated, partially unsaturated, or aryl ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. Suitabledivalent substituents that are bound to vicinal substitutable carbons ofan “optionally substituted” group include: —O(CR^(*) ₂)₂₋₃O—, whereineach independent occurrence of R^(*) is selected from hydrogen, C₁₋₆aliphatic which may be substituted as defined below, or an unsubstituted5-6-membered saturated, partially unsaturated, or aryl ring having 0-4heteroatoms independently selected from nitrogen, oxygen, or sulfur.

Suitable substituents on the aliphatic group of R^(*) include halogen,—R^(), -(haloR^()), —OH, —OR^(), —O(haloR^()), —CN, —C(O)OH,—C(O)OR^(), —NH₂, —NHR^(), —NR^() ₂, or —NO₂, wherein each R^() isunsubstituted or where preceded by “halo” is substituted only with oneor more halogens, and is independently C₁₋₄ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur.

Suitable substituents on a substitutable nitrogen of an “optionallysubstituted” group include —R^(†), —NR^(†) ₂, —C(O)R^(†), —C(O)OR^(†),—C(O)C(O)R^(†), —C(O)CH₂C(O)R^(†), —S(O)₂R^(†), —S(O)₂NR^(†) ₂,—C(S)NR^(†) ₂, —C(NH)NR^(†) ₂, or —N(R^(†))S(O)₂R^(†); wherein eachR^(†)is independently hydrogen, C₁₋₆ aliphatic which may be substitutedas defined below, unsubstituted —OPh, or an unsubstituted 5-6-memberedsaturated, partially unsaturated, or aryl ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, or,notwithstanding the definition above, two independent occurrences ofR^(†), taken together with their intervening atom(s) form anunsubstituted 3-12-membered saturated, partially unsaturated, or arylmono- or bicyclic ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur.

Suitable substituents on the aliphatic group of R^(†)are independentlyhalogen, —R^(), -(haloR^()), —OH, —OR′, —O(haloR^()), —CN, —C(O)OH,—C(O)OR^(), —NH₂, —NHR^(), —NR^() ₂, or —NO₂, wherein each R^() isunsubstituted or where preceded by “halo” is substituted only with oneor more halogens, and is independently C₁₋₄ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur.

As used herein, the term “pharmaceutically acceptable salt” refers tothose salts which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswithout undue toxicity, irritation, allergic response and the like, andare commensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts are well known in the art. For example, S. M. Berge etal., describe pharmaceutically acceptable salts in detail in J.Pharmaceutical Sciences, 1977, 66, 1-19, incorporated herein byreference. Pharmaceutically acceptable salts of the compounds of thisinvention include those derived from suitable inorganic and organicacids and bases. Examples of pharmaceutically acceptable, nontoxic acidaddition salts are salts of an amino group formed with inorganic acidssuch as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuricacid and perchloric acid or with organic acids such as acetic acid,oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid ormalonic acid or by using other methods used in the art such as ionexchange. Other pharmaceutically acceptable salts include adipate,alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate,borate, butyrate, camphorate, camphorsulfonate, citrate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,formate, fumarate, glucoheptonate, glycerophosphate, gluconate,hemisulfate, heptanoate, hexanoate, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, pivalate,propionate, stearate, succinate, sulfate, tartrate, thiocyanate,p-toluenesulfonate, undecanoate, valerate salts, and the like.

Salts derived from appropriate bases include alkali metal, alkalineearth metal, ammonium and N^(|)(C₁₋₄alkyl)₄ salts. Representative alkalior alkaline earth metal salts include sodium, lithium, potassium,calcium, magnesium, and the like. Further pharmaceutically acceptablesalts include, when appropriate, nontoxic ammonium, quaternary ammonium,and amine cations formed using counterions such as halide, hydroxide,carboxylate, sulfate, phosphate, nitrate, loweralkyl sulfonate and arylsulfonate.

Unless otherwise stated, structures depicted herein are also meant toinclude all isomeric (e.g., enantiomeric, diastereomeric, and geometric(or conformational)) forms of the structure; for example, the R and Sconfigurations for each asymmetric center, Z and E double bond isomers,and Z and E conformational isomers. Therefore, single stereochemicalisomers as well as enantiomeric, diastereomeric, and geometric (orconformational) mixtures of the present compounds are within the scopeof the invention. Unless otherwise stated, all tautomeric forms of thecompounds of the invention are within the scope of the invention.Additionally, unless otherwise stated, structures depicted herein arealso meant to include compounds that differ only in the presence of oneor more isotopically enriched atoms. For example, compounds having thepresent structures including the replacement of hydrogen by deuterium ortritium, or the replacement of a carbon by ¹³C- or ¹⁴C-enriched carbonare within the scope of this invention. Such compounds are useful, forexample, as analytical tools, as probes in biological assays, or astherapeutic agents in accordance with the present invention. In certainembodiments, a warhead moiety, R³, of a provided compound comprises oneor more deuterium atoms.

As used herein, the term “irreversible” or “irreversible inhibitor”refers to an inhibitor (i.e. a compound) that is able to be covalentlybonded to HCV protease in a substantially non-reversible manner. Thatis, whereas a reversible inhibitor is able to bind to (but is generallyunable to form a covalent bond with) HCV protease, and therefore canbecome dissociated from the HCV protease an irreversible inhibitor willremain substantially bound to HCV protease once covalent bond formationhas occurred. Irreversible inhibitors usually display time dependency,whereby the degree of inhibition increases with the time with which theinhibitor is in contact with the enzyme. In certain embodiments, anirreversible inhibitor will remain substantially bound to HCV proteaseonce covalent bond formation has occurred and will remain bound for atime period that is longer than the life of the protein.

Methods for identifying if a compound is acting as an irreversibleinhibitor are known to one of ordinary skill in the art. Such methodsinclude, but are not limited to, enzyme kinetic analysis of theinhibition profile of the compound with HCV protease, the use of massspectrometry of the protein drug target modified in the presence of theinhibitor compound, discontinuous exposure, also known as “washout,”experiments, and the use of labeling, such as radiolabelled inhibitor,to show covalent modification of the enzyme, as well as other methodsknown to one of skill in the art.

One of ordinary skill in the art will recognize that certain reactivefunctional groups can act as “warheads.” As used herein, the term“warhead” or “warhead group” refers to a functional group present on acompound of the present invention wherein that functional group iscapable of covalently binding to an amino acid residue (such ascysteine, lysine, histidine, or other residues capable of beingcovalently modified) present in the binding pocket of the targetprotein, thereby irreversibly inhibiting the protein. It will beappreciated that the -L-Y group, as defined and described herein,provides such warhead groups for covalently, and irreversibly,inhibiting the protein.

As used herein, the term “inhibitor” is defined as a compound that bindsto and/or inhibits HCV protease with measurable affinity. In certainembodiments, an inhibitor has an IC₅₀ and/or binding constant of lessabout 50 μM, less than about 1 μM, less than about 500 nM, less thanabout 100 nM, less than about 10 nM, or less than about 1 nM.

A compound of the present invention may be tethered to a detectablemoiety. One of ordinary skill in the art will recognize that adetectable moiety may be attached to a provided compound via a suitablesubstituent. As used herein, the term “suitable substituent” refers to amoiety that is capable of covalent attachment to a detectable moiety.Such moieties are well known to one of ordinary skill in the art andinclude groups containing, e.g., a carboxylate moiety, an amino moiety,a thiol moiety, or a hydroxyl moiety, to name but a few. It will beappreciated that such moieties may be directly attached to a providedcompound or via a tethering group, such as a bivalent saturated orunsaturated hydrocarbon chain. In some embodiments, such moieties may beattached via click chemistry. In some embodiments, such moieties may beattached via a 1,3-cycloaddition of an azide with an alkyne, optionallyin the presence of a copper catalyst. Methods of using click chemistryare known in the art and include those described by Rostovtsev et al.,Angew. Chem. Int. Ed. 2002, 41, 2596-99 and Sun et al., BioconjugateChem., 2006, 17, 52-57.

As used herein, the term “detectable moiety” is used interchangeablywith the term “label” and relates to any moiety capable of beingdetected, e.g., primary labels and secondary labels. Primary labels,such as radioisotopes (e.g., tritium, ³²P, ³³P, ³⁵S, or ¹⁴C), mass-tags,and fluorescent labels are signal generating reporter groups which canbe detected without further modifications. Detectable moieties alsoinclude luminescent and phosphorescent groups.

The term “secondary label” as used herein refers to moieties such asbiotin and various protein antigens that require the presence of asecond intermediate for production of a detectable signal. For biotin,the secondary intermediate may include streptavidin-enzyme conjugates.For antigen labels, secondary intermediates may include antibody-enzymeconjugates. Some fluorescent groups act as secondary labels because theytransfer energy to another group in the process of nonradiativefluorescent resonance energy transfer (FRET), and the second groupproduces the detected signal.

The terms “fluorescent label”, “fluorescent dye”, and “fluorophore” asused herein refer to moieties that absorb light energy at a definedexcitation wavelength and emit light energy at a different wavelength.Examples of fluorescent labels include, but are not limited to: AlexaFluor dyes (Alexa Fluor 350, Alexa Fluor 488, Alexa Fluor 532, AlexaFluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, AlexaFluor 660 and Alexa Fluor 680), AMCA, AMCA-S, BODIPY dyes (BODIPY FL,BODIPY R6G, BODIPY TMR, BODIPY TR, BODIPY 530/550, BODIPY 558/568,BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY 630/650, BODIPY650/665), Carboxyrhodamine 6G, carboxy-X-rhodamine (ROX), Cascade Blue,Cascade Yellow, Coumarin 343, Cyanine dyes (Cy3, Cy5, Cy3.5, Cy5.5),Dansyl, Dapoxyl, Dialkylaminocoumarin,4′,5′-Dichloro-2′,7′-dimethoxy-fluorescein, DM-NERF, Eosin, Erythrosin,Fluorescein, FAM, Hydroxycoumarin, IRDyes (IRD40, IRD 700, IRD 800),JOE, Lissamine rhodamine B, Marina Blue, Methoxycoumarin,Naphthofluorescein, Oregon Green 488, Oregon Green 500, Oregon Green514, Pacific Blue, PyMPO, Pyrene, Rhodamine B, Rhodamine 6G, RhodamineGreen, Rhodamine Red, Rhodol Green,2′,4′,5′,7′-Tetra-bromosulfone-fluorescein, Tetramethyl-rhodamine (TMR),Carboxytetramethylrhodamine (TAMRA), Texas Red, Texas Red-X.

The term “mass-tag” as used herein refers to any moiety that is capableof being uniquely detected by virtue of its mass using mass spectrometry(MS) detection techniques. Examples of mass-tags include electrophorerelease tags such asN-[3-[4′-[(p-Methoxytetrafluorobenzyl)oxy]phenyl]-3-methylglyceronyl]isonipecoticAcid, 4′-[2,3,5,6-Tetrafluoro-4-(pentafluorophenoxyl)]methylacetophenone, and their derivatives. The synthesis and utility of thesemass-tags is described in U.S. Pat. Nos. 4,650,750, 4,709,016,5,360,8191, 5,516,931, 5,602,273, 5,604,104, 5,610,020, and 5,650,270.Other examples of mass-tags include, but are not limited to,nucleotides, dideoxynucleotides, oligonucleotides of varying length andbase composition, oligopeptides, oligosaccharides, and other syntheticpolymers of varying length and monomer composition. A large variety oforganic molecules, both neutral and charged (biomolecules or syntheticcompounds) of an appropriate mass range (100-2000 Daltons) may also beused as mass-tags.

The terms “measurable affinity” and “measurably inhibit,” as usedherein, means a measurable change in HCV protease activity between asample comprising a compound of the present invention, or compositionthereof, and HCV protease, and an equivalent sample comprising HCVprotease, in the absence of said compound, or composition thereof.

3. Description of Exemplary Compounds

In certain embodiments, the present invention provides a compound offormula I:

or a pharmaceutically acceptable salt thereof, wherein:

-   R¹ and R^(1′) are independently hydrogen or optionally substituted    C₁₋₆ aliphatic, or R¹ and R^(1′) are taken together to form an    optionally substituted 3-7 membered carbocyclic ring;-   R^(2a) is —OH or —NHSO₂R²;-   R² is —N(R)₂ or an optionally substituted group selected from C₃₋₇    cycloalkyl, a bridged bicyclic, 6-10 membered aryl, 5-10 membered    heteroaryl having 1-4 heteroatoms independently selected from    nitrogen, oxygen, or sulfur, or 4-7 membered heterocyclyl having 1-2    heteroatoms independently selected from nitrogen, oxygen, or sulfur;-   each R is independently hydrogen, optionally substituted C₁₋₆    aliphatic, or:    -   two R on the same nitrogen atom are taken together with the        nitrogen to form a 4-7 membered heterocyclic ring having 1-2        heteroatoms independently selected from nitrogen, oxygen, or        sulfur;-   R³ is -L-Y, wherein:    -   L is a covalent bond or a bivalent C₁₋₈ saturated or        unsaturated, straight or branched, hydrocarbon chain, wherein        one, two, or three methylene units of L are optionally and        independently replaced by cyclopropylene, —NR—, —N(R)C(O)—,        —C(O)N(R)—, —N(R)SO₂—, —SO₂N(R)—, —O—, —C(O)—, —OC(O)—, —C(O)O—,        —S—, —SO—, —SO₂—, —C(═S)—, —C(═NR)—, —N═N—, or —C(═N₂)—;    -   Y is hydrogen, C₁₋₆ aliphatic optionally substituted with oxo,        halogen, NO₂, or CN, or a 3-10 membered monocyclic or bicyclic,        saturated, partially unsaturated, or aryl ring having 0-3        heteroatoms independently selected from nitrogen, oxygen, or        sulfur, and wherein said ring is substituted with 1-4 R^(e)        groups; and    -   each R^(e) is independently selected from -Q-Z, oxo, NO₂,        halogen, CN, a suitable leaving group, or a C₁₋₆ aliphatic        optionally substituted with oxo, halogen, NO₂, or CN, wherein:        -   Q is a covalent bond or a bivalent C₁₋₆ saturated or            unsaturated, straight or branched, hydrocarbon chain,            wherein one or two methylene units of Q are optionally and            independently replaced by —N(R)—, —S—, —O—, —C(O)—, —OC(O)—,            —C(O)O—, —SO—, or —SO₂—, —N(R)C(O)—, —C(O)N(R)—, —N(R)SO₂—,            or —SO₂N(R)—; and    -   Z is hydrogen or C₁₋₆ aliphatic optionally substituted with oxo,        halogen, NO₂, or CN; or    -   R³ and a ring formed by R¹ and R^(1′) are taken together with        their intervening atoms to form an optionally substituted        saturated or unsaturated 12-18 membered ring having 2-6        heteroatoms independently selected from nitrogen oxygen, or        sulfur, wherein the ring formed thereby comprises a warhead        group;-   R⁴ is H, —NHC(O)R⁵, —NHC(O)OR⁶,

or a natural or unnatural amino acid side-chain group;

-   each R⁵ is independently —N(R)₂ or an optionally substituted group    selected from C₁₋₆ aliphatic, a bridged bicyclic, 6-10 membered    aryl, 5-10 membered heteroaryl having 1-4 heteroatoms independently    selected from nitrogen, oxygen, or sulfur, or 4-7 membered    heterocyclyl having 1-2 heteroatoms independently selected from    nitrogen, oxygen, or sulfur;-   R⁶ is an optionally substituted group selected from C₁₋₆ aliphatic,    a bridged bicyclic, 6-10 membered aryl, 5-10 membered heteroaryl    having 1-4 heteroatoms independently selected from nitrogen, oxygen,    or sulfur, or 4-7 membered heterocyclyl having 1-2 heteroatoms    independently selected from nitrogen, oxygen, or sulfur; and-   R⁷ is an optionally substituted group selected from C₁₋₆ aliphatic,    a bridged bicyclic, 6-10 membered aryl, 5-10 membered heteroaryl    having 1-4 heteroatoms independently selected from nitrogen, oxygen,    or sulfur, or 4-7 membered heterocyclyl having 1-2 heteroatoms    independently selected from nitrogen, oxygen, or sulfur; and-   R^(z) is

or R⁴ and R^(z) are taken together with their intervening atoms to forman optionally substituted, saturated or unsaturated 16-22 membered ringhaving 2-6 heteroatoms independently selected from nitrogen, oxygen, orsulfur; each occurrence of R^(y) is independently selected from halogen,—OR^(◯), —CN, —NO₂, —N(R^(◯))₂, or optionally substituted C₁₋₄aliphatic; and

-   m is an integer from 0 to 4, inclusive;-   s is an integer from 0 to 4, inclusive;-   t is an integer from 0 to 4, inclusive;    wherein the sum of s and t is non-zero.

In certain embodiments, L is a covalent bond.

In certain embodiments, L is a bivalent C₁₋₈ saturated or unsaturated,straight or branched, hydrocarbon chain, wherein one, two, or threemethylene units of L are optionally and independently replaced bycyclopropylene, —NR—, —N(R)C(O)—, —C(O)N(R)—, —N(R)SO₂—, —SO₂N(R)—, —O—,—C(O)—, —OC(O)—, —C(O)O—, —S—, —SO—, —SO₂—, —C(═S)—, —C(═NR)—, —N═N—, or—C(═N₂)—.

In certain embodiments, L is a bivalent C₁₋₈ saturated or unsaturated,straight or branched, hydrocarbon chain. In certain embodiments, L is—CH₂—.

In certain embodiments, L is a covalent bond, —CH₂—, —NH—, —CH₂NH—,—NHCH₂—, —NHC(O)—, —NHC(O)CH₂OC(O)—, —CH₂NHC(O)—, —NHSO₂—, —NHSO₂CH₂—,—NHC(O)CH₂OC(O)—, or —SO₂NH—.

In some embodiments, L is a bivalent C₂₋₈ straight or branched,hydrocarbon chain wherein L has at least one double bond and one or twoadditional methylene units of L are optionally and independentlyreplaced by —NRC(O)—, —C(O)NR—, —N(R)SO₂—, —SO₂N(R)—, —S—, —S(O)—,—SO₂—, —OC(O)—, —C(O)O—, —O—, —N(R)—, or —C(O)—.

In certain embodiments, L is a bivalent C₂₋₈ straight or branched,hydrocarbon chain wherein L has at least one double bond and at leastone methylene unit of L is replaced by —C(O)—, —NRC(O)—, —C(O)NR—,—N(R)SO₂—, —SO₂N(R)—, —S—, —S(O)—, —SO₂—, —OC(O)—, or —C(O)O—, and oneor two additional methylene units of L are optionally and independentlyreplaced by cyclopropylene, —O—, —N(R)—, or —C(O)—.

In some embodiments, L is a bivalent C₂₋₈ straight or branched,hydrocarbon chain wherein L has at least one double bond and at leastone methylene unit of L is replaced by —C(O)—, and one or two additionalmethylene units of L are optionally and independently replaced bycyclopropylene, —O—, —N(R)—, or —C(O)—.

As described above, in certain embodiments, L is a bivalent C₂₋₈straight or branched, hydrocarbon chain wherein L has at least onedouble bond. One of ordinary skill in the art will recognize that such adouble bond may exist within the hydrocarbon chain backbone or may be“exo” to the backbone chain and thus forming an alkylidene group. By wayof example, such an L group having an alkylidene branched chain includes—CH₂C(═CH₂)CH₂—. Thus, in some embodiments, L is a bivalent C₂₋₈straight or branched, hydrocarbon chain wherein L has at least onealkylidenyl double bond. Exemplary L groups include —NHC(O)C(═CH₂)CH₂—.

In certain embodiments, L is a bivalent C₂₋₈ straight or branched,hydrocarbon chain wherein L has at least one double bond and at leastone methylene unit of L is replaced by —C(O)—. In certain embodiments, Lis —C(O)CH═CH(CH₃)—, —C(O)CH═CHCH₂NH(CH₃)—, —C(O)CH═CH(CH₃)—,—C(O)CH═CH—, —CH₂C(O)CH═CH—, —CH₂C(O)CH═CH(CH₃)—, —CH₂CH₂C(O)CH═CH—,—CH₂CH₂C(O)CH═CHCH₂—, —CH₂CH₂C(O)CH═CHCH₂NH(CH₃)—, or—CH₂CH₂C(O)CH═CH(CH₃)—, or —CH(CH₃)OC(O)CH═CH—.

In certain embodiments, L is a bivalent C₂₋₈ straight or branched,hydrocarbon chain wherein L has at least one double bond and at leastone methylene unit of L is replaced by —OC(O)—.

In some embodiments, L is a bivalent C₂₋₈ straight or branched,hydrocarbon chain wherein L has at least one double bond and at leastone methylene unit of L is replaced by —NRC(O)—, —C(O)NR—, —N(R)SO₂—,—SO₂N(R)—, —S—, —S(O)—, —SO₂—, —OC(O)—, or —C(O)O—, and one or twoadditional methylene units of L are optionally and independentlyreplaced by cyclopropylene, —O—, —N(R)—, or —C(O)—. In some embodiments,L is —CH₂OC(O)CH═CHCH₂—, —CH₂—OC(O)CH═CH—, or —CH(CH═CH₂)OC(O)CH═CH—.

In certain embodiments, L is —NRC(O)CH═CH—, —NRC(O)CH═CHCH₂N(CH₃)—,—NRC(O)CH═CHCH₂O—, —CH₂NRC(O)CH═CH—, —NRSO₂CH═CH—, —NRSO₂CH═CHCH₂—,—NRC(O)(C═N₂)C(O)—, —NRC(O)CH═CHCH₂N(CH₃)—, —NRSO₂CH═CH—,—NRSO₂CH═CHCH₂—, —NRC(O)CH═CHCH₂O—, —NRC(O)C(═CH₂)CH₂—, —CH₂NRC(O)—,—CH₂NRC(O)CH═CH—, —CH₂CH₂NRC(O)—, or —CH₂NRC(O)cyclopropylene-, whereineach R is independently hydrogen or optionally substituted C₁₋₆aliphatic.

In certain embodiments, L is —NHC(O)CH═CH—, —NHC(O)CH═CHCH₂N(CH₃)—,—NHC(O)CH═CHCH₂O—, —CH₂NHC(O)CH═CH—, —NHSO₂CH═CH—, —NHSO₂CH═CHCH₂—,—NHC(O)(C═N₂)C(O)—, —NHC(O)CH═CHCH₂N(CH₃)—, —NHSO₂CH═CH—,—NHSO₂CH═CHCH₂—, —NHC(O)CH═CHCH₂O—, —NHC(O)C(═CH₂)CH₂—, —CH₂NHC(O)—,—CH₂NHC(O)CH═CH—, —CH₂CH₂NHC(O)—, or —CH₂NHC(O)cyclopropylene-.

In some embodiments, L is a bivalent C₂₋₈ straight or branched,hydrocarbon chain wherein L has at least one triple bond. In certainembodiments, L is a bivalent C₂₋₈ straight or branched, hydrocarbonchain wherein L has at least one triple bond and one or two additionalmethylene units of L are optionally and independently replaced by—NRC(O)—, —C(O)NR—, —S—, —S(O)—, —SO₂—, —C(═S)—, —C(═NR)—, —O—, —N(R)—,or —C(O)—. In some embodiments, L has at least one triple bond and atleast one methylene unit of L is replaced by —N(R)—, —N(R)C(O)—, —C(O)—,—C(O)O—, or —OC(O)—, or —O—.

Exemplary L groups include —C≡C—, —C≡CCH₂N(isopropyl)-,—NHC(O)C≡CCH₂CH₂—, —CH₂—C≡C—CH₂—, —C≡CCH₂O—, —CH₂C(O)C≡C—, —C(O)C≡C—, or—CH₂C(═O)C≡C—.

In certain embodiments, L is a bivalent C₂₋₈ straight or branched,hydrocarbon chain wherein one methylene unit of L is replaced bycyclopropylene and one or two additional methylene units of L areindependently replaced by —C(O)—, —NRC(O)—, —C(O)NR—, —N(R)SO₂—, or—SO₂N(R)—. Exemplary L groups include —NHC(O)-cyclopropylene-SO₂— and—NHC(O)-cyclopropylene-.

As defined generally above, Y is hydrogen, C₁₋₆ aliphatic optionallysubstituted with oxo, halogen, NO₂, or CN, or a 3-10 membered monocyclicor bicyclic, saturated, partially unsaturated, or aryl ring having 0-3heteroatoms independently selected from nitrogen, oxygen, or sulfur, andwherein said ring is substituted with at 1-4 R^(e) groups, each R^(e) isindependently selected from -Q-Z, oxo, NO₂, halogen, CN, a suitableleaving group, or C₁₋₆ aliphatic, wherein Q is a covalent bond or abivalent C₁₋₆ saturated or unsaturated, straight or branched,hydrocarbon chain, wherein one or two methylene units of Q areoptionally and independently replaced by —N(R)—, —S—, —O—, —C(O)—,—OC(O)—, —C(O)O—, —SO—, or —SO₂—, —N(R)C(O)—, —C(O)N(R)—, —N(R)SO₂—, or—SO₂N(R)—; and, Z is hydrogen or C₁₋₆ aliphatic optionally substitutedwith oxo, halogen, NO₂, or CN.

In certain embodiments, Y is hydrogen.

In certain embodiments, Y is C₁₋₆ aliphatic optionally substituted withoxo, halogen, NO₂, or CN. In some embodiments, Y is C₂₋₆ alkenyloptionally substituted with oxo, halogen, NO₂, or CN. In otherembodiments, Y is C₂₋₆ alkynyl optionally substituted with oxo, halogen,NO₂, or CN. In some embodiments, Y is C₂₋₆ alkenyl. In otherembodiments, Y is C₂₋₄ alkynyl.

In other embodiments, Y is C₁₋₆ alkyl substituted with oxo, halogen,NO₂, or CN. Such Y groups include —CH₂F, —CH₂Cl, —CH₂CN, and —CH₂NO₂.

In certain embodiments, Y is a saturated 3-6 membered monocyclic ringhaving 0-3 heteroatoms independently selected from nitrogen, oxygen, orsulfur, wherein Y is substituted with 1-4 R^(e) groups, wherein eachR^(e) is as defined above and described herein.

In some embodiments, Y is a saturated 3-4 membered heterocyclic ringhaving 1 heteroatom selected from oxygen or nitrogen wherein said ringis substituted with 1-2 R^(e) groups, wherein each R^(e) is as definedabove and described herein. Exemplary such rings are epoxide and oxetanerings, wherein each ring is substituted with 1-2 R^(e) groups, whereineach R^(e) is as defined above and described herein.

In other embodiments, Y is a saturated 5-6 membered heterocyclic ringhaving 1-2 heteroatom selected from oxygen or nitrogen wherein said ringis substituted with 1-4 R^(e) groups, wherein each R^(e) is as definedabove and described herein. Such rings include piperidine andpyrrolidine, wherein each ring is substituted with 1-4 R^(e) groups,wherein each R^(e) is as defined above and described herein. In certainembodiments, Y is

wherein each R, Q, Z, and R^(e) is as defined above and describedherein.

In some embodiments, Y is a saturated 3-6 membered carbocyclic ring,wherein said ring is substituted with 1-4 R^(e) groups, wherein eachR^(e) is as defined above and described herein. In certain embodiments,Y is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl, wherein eachring is substituted with 1-4 R^(e) groups, wherein each R^(e) is asdefined above and described herein. In certain embodiments, Y is

wherein R^(e) is as defined above and described herein. In certainembodiments, Y is cyclopropyl optionally substituted with halogen, CN orNO₂.

In certain embodiments, Y is a partially unsaturated 3-6 memberedmonocyclic ring having 0-3 heteroatoms independently selected fromnitrogen, oxygen, or sulfur, wherein said ring is substituted with 1-4R^(e) groups, wherein each R^(e) is as defined above and describedherein.

In some embodiments, Y is a partially unsaturated 3-6 memberedcarbocyclic ring, wherein said ring is substituted with 1-4 R^(e)groups, wherein each R^(e) is as defined above and described herein. Insome embodiments, Y is cyclopropenyl, cyclobutenyl, cyclopentenyl, orcyclohexenyl wherein each ring is substituted with 1-4 R^(e) groups,wherein each R^(e) is as defined above and described herein. In certainembodiments, Y is

wherein each R^(e) is as defined above and described herein.

In certain embodiments, Y is a partially unsaturated 4-6 memberedheterocyclic ring having 1-2 heteroatoms independently selected fromnitrogen, oxygen, or sulfur, wherein said ring is substituted with 1-4R^(e) groups, wherein each R^(e) is as defined above and describedherein. In certain embodiments, Y is selected from:

wherein each R and R^(e) is as defined above and described herein.

In certain embodiments, Y is a 6-membered aromatic ring having 0-2nitrogens wherein said ring is substituted with 1-4 R^(e) groups,wherein each R^(e) group is as defined above and described herein. Incertain embodiments, Y is phenyl, pyridyl, or pyrimidinyl, wherein eachring is substituted with 1-4 R^(e) groups, wherein each R^(e) is asdefined above and described herein.

In some embodiments, Y is selected from:

wherein each R^(e) is as defined above and described herein.

In other embodiments, Y is a 5-membered heteroaryl ring having 1-3heteroatoms independently selected from nitrogen, oxygen, or sulfur,wherein said ring is substituted with 1-3 R^(e) groups, wherein eachR^(e) group is as defined above and described herein. In someembodiments, Y is a 5 membered partially unsaturated or aryl ring having1-3 heteroatoms independently selected from nitrogen, oxygen, andsulfur, wherein said ring is substituted with 1-4 R^(e) groups, whereineach R^(e) group is as defined above and described herein. Exemplarysuch rings are isoxazolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl,pyrrolyl, furanyl, thienyl, triazole, thiadiazole, and oxadiazole,wherein each ring is substituted with 1-3 R^(e) groups, wherein eachR^(e) group is as defined above and described herein. In certainembodiments, Y is selected from:

wherein each R and R^(e) is as defined above and described herein.

In certain embodiments, Y is an 8-10 membered bicyclic, saturated,partially unsaturated, or aryl ring having 0-3 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur, wherein said ring issubstituted with 1-4 R^(e) groups, wherein R^(e) is as defined above anddescribed herein. According to another aspect, Y is a 9-10 memberedbicyclic, partially unsaturated, or aryl ring having 1-3 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, wherein saidring is substituted with 1-4 R^(e) groups, wherein R^(e) is as definedabove and described herein. Exemplary such bicyclic rings include2,3-dihydrobenzo[d]isothiazole, wherein said ring is substituted with1-4 R^(e) groups, wherein R^(e) is as defined above and describedherein.

As defined generally above, each R^(e) group is independently selectedfrom -Q-Z, oxo, NO₂, halogen, CN, a suitable leaving group, or C₁₋₆aliphatic optionally substituted with oxo, halogen, NO₂, or CN, whereinQ is a covalent bond or a bivalent C₁₋₆ saturated or unsaturated,straight or branched, hydrocarbon chain, wherein one or two methyleneunits of Q are optionally and independently replaced by —N(R)—, —S—,—O—, —C(O)—, —OC(O)—, —C(O)O—, —SO—, or —SO₂—, —N(R)C(O)—, —C(O)N(R)—,—N(R)SO₂—, or —SO₂N(R)—; and Z is hydrogen or C₁₋₆ aliphatic optionallysubstituted with oxo, halogen, NO₂, or CN.

In certain embodiments, R^(e) is C₁₋₆ aliphatic optionally substitutedwith oxo, halogen, NO₂, or CN. In other embodiments, R^(e) is oxo, NO₂,halogen, or CN.

In some embodiments, R^(e) is -Q-Z, wherein Q is a covalent bond and Zis hydrogen (i.e., R^(e) is hydrogen). In other embodiments, R^(e) is-Q-Z, wherein Q is a bivalent C₁₋₆ saturated or unsaturated, straight orbranched, hydrocarbon chain, wherein one or two methylene units of Q areoptionally and independently replaced by —NR—, —NRC(O)—, —C(O)NR—, —S—,—O—, —C(O)—, —SO—, or —SO₂—. In other embodiments, Q is a bivalent C₂₋₆straight or branched, hydrocarbon chain having at least one double bond,wherein one or two methylene units of Q are optionally and independentlyreplaced by —NR—, —NRC(O)—, —C(O)NR—, —S—, —O—, —C(O)—, —SO—, or —SO₂—.In certain embodiments, the Z moiety of the R^(e) group is hydrogen. Insome embodiments, -Q-Z is —NHC(O)CH═CH₂ or —C(O)CH═CH₂.

In certain embodiments, each R^(e) is independently selected from oxo,NO₂, CN, fluoro, chloro, —NHC(O)CH═CH₂, —C(O)CH═CH₂, —CH₂CH═CH₂,—C(O)OCH₂Cl, —C(O)OCH₂F, —C(O)OCH₂CN, —C(O)CH₂Cl, —C(O)CH₂F, —C(O)CH₂CN,or —CH₂C(O)CH₃.

In certain embodiments, R^(e) is a suitable leaving group, ie a groupthat is subject to nucleophilic displacement. A “suitable leaving” is achemical group that is readily displaced by a desired incoming chemicalmoiety such as the thiol moiety of a cysteine of interest. Suitableleaving groups are well known in the art, e.g., sec, “Advanced OrganicChemistry,” Jerry March, 5^(th) Ed., pp. 351-357, John Wiley and Sons,N.Y. Such leaving groups include, but are not limited to, halogen,alkoxy, sulphonyloxy, optionally substituted alkylsulphonyloxy,optionally substituted alkenylsulfonyloxy, optionally substitutedarylsulfonyloxy, acyl, and diazonium moieties. Examples of suitableleaving groups include chloro, iodo, bromo, fluoro, acetoxy,methanesulfonyloxy (mesyloxy), tosyloxy, triflyloxy,nitro-phenylsulfonyloxy (nosyloxy), and bromo-phenylsulfonyloxy(brosyloxy).

In certain embodiments, the following embodiments and combinations of-L-Y apply:

-   -   (a) L is a bivalent C₂₋₈ straight or branched, hydrocarbon chain        wherein L has at least one double bond and one or two additional        methylene units of L are optionally and independently replaced        by —NRC(O)—, —C(O)NR—, —N(R)SO₂—, —SO₂N(R)—, —S—, —S(O)—, —SO₂—,        —OC(O)—, —C(O)O—, cyclopropylene, —O—, —N(R)—, or —C(O)—; and Y        is hydrogen or C₁₋₆ aliphatic optionally substituted with oxo,        halogen, NO₂, or CN; or    -   (b) L is a bivalent C₂₋₈ straight or branched, hydrocarbon chain        wherein L has at least one double bond and at least one        methylene unit of L is replaced by —C(O)—, —NRC(O)—, —C(O)NR—,        —N(R)SO₂—, —SO₂N(R)—, —S—, —S(O)—, —SO₂—, —OC(O)—, or —C(O)O—,        and one or two additional methylene units of L are optionally        and independently replaced by cyclopropylene, —O—, —N(R)—, or        —C(O)—; and Y is hydrogen or C₁₋₆ aliphatic optionally        substituted with oxo, halogen, NO₂, or CN; or    -   (c) L is a bivalent C₂₋₈ straight or branched, hydrocarbon chain        wherein L has at least one double bond and at least one        methylene unit of L is replaced by —C(O)—, and one or two        additional methylene units of L are optionally and independently        replaced by cyclopropylene, —O—, —N(R)—, or —C(O)—; and Y is        hydrogen or C₁₋₆ aliphatic optionally substituted with oxo,        halogen, NO₂, or CN; or    -   (d) L is a bivalent C₂₋₈ straight or branched, hydrocarbon chain        wherein L has at least one double bond and at least one        methylene unit of L is replaced by —C(O)—; and Y is hydrogen or        C₁₋₆ aliphatic optionally substituted with oxo, halogen, NO₂, or        CN; or    -   (e) L is a bivalent C₂₋₈ straight or branched, hydrocarbon chain        wherein L has at least one double bond and at least one        methylene unit of L is replaced by —OC(O)—; and Y is hydrogen or        C₁₋₆ aliphatic optionally substituted with oxo, halogen, NO₂, or        CN; or    -   (f) L is —NRC(O)CH═CH—, —NRC(O)CH═CHCH₂N(CH₃)—,        —NRC(O)CH═CHCH₂O—, —CH₂NRC(O)CH═CH—, —NRSO₂CH═CH—,        —NRSO₂CH═CHCH₂—, —NRC(O)(C═N₂)—, —NRC(O)(C═N₂)C(O)—,        —NRC(O)CH═CHCH₂N(CH₃)—, —NRSO₂CH═CH—, —NRSO₂CH═CHCH₂—,        —NRC(O)CH═CHCH₂O—, —NRC(O)C(═CH₂)CH₂—, —CH₂NRC(O)—,        —CH₂NRC(O)CH═CH—, —CH₂CH₂NRC(O)—, or —CH₂NRC(O)cyclopropylene-;        wherein R is H or optionally substituted C₁₋₆ aliphatic; and Y        is hydrogen or C₁₋₆ aliphatic optionally substituted with oxo,        halogen, NO₂, or CN; or    -   (g) L is —NHC(O)CH═CH—, —NHC(O)CH═CHCH₂N(CH₃)—,        —NHC(O)CH═CHCH₂O—, —CH₂NHC(O)CH═CH—, —NHSO₂CH═CH—,        —NHSO₂CH═CHCH₂—, —NHC(O)(C═N₂)—, —NHC(O)(C═N₂)C(O)—,        —NHC(O)CH═CHCH₂N(CH₃)—, —NHSO₂CH═CH—, —NHSO₂CH═CHCH₂—,        —NHC(O)CH═CHCH₂O—, —NHC(O)C(═CH₂)CH₂—, —CH₂NHC(O)—,        —CH₂NHC(O)CH═CH—, —CH₂CH₂NHC(O)—, or —CH₂NHC(O)cyclopropylene-;        and Y is hydrogen or C₁₋₆ aliphatic optionally substituted with        oxo, halogen, NO₂, or CN; or    -   (h) L is a bivalent C₂₋₈ straight or branched, hydrocarbon chain        wherein L has at least one alkylidenyl double bond and at least        one methylene unit of L is replaced by —C(O)—, —NRC(O)—,        —C(O)NR—, —N(R)SO₂—, —SO₂N(R)—, —S—, —S(O)—, —SO₂—, —OC(O)—, or        —C(O)O—, and one or two additional methylene units of L are        optionally and independently replaced by cyclopropylene, —O—,        —N(R)—, or —C(O)—; and Y is hydrogen or C₁₋₆ aliphatic        optionally substituted with oxo, halogen, NO₂, or CN; or    -   (i) L is a bivalent C₂₋₈ straight or branched, hydrocarbon chain        wherein L has at least one triple bond and one or two additional        methylene units of L are optionally and independently replaced        by —NRC(O)—, —C(O)NR—, —N(R)SO₂—, —SO₂N(R)—, —S—, —S(O)—, —SO₂—,        —OC(O)—, or —C(O)O—, and Y is hydrogen or C₁₋₆ aliphatic        optionally substituted with oxo, halogen, NO₂, or CN; or    -   (j) L is —C≡C—, —C≡CCH₂N(isopropyl)-, —NHC(O)C≡CCH₂CH₂—,        —CH₂—C≡C—CH₂—, —C≡CCH₂O—, —CH₂C(O)C≡C—, —C(O)C≡C—, or        —CH₂OC(═O)C≡C—; and Y is hydrogen or C₁₋₆ aliphatic optionally        substituted with oxo, halogen, NO₂, or CN; or    -   (k) L is a bivalent C₂₋₈ straight or branched, hydrocarbon chain        wherein one methylene unit of L is replaced by cyclopropylene        and one or two additional methylene units of L are independently        replaced by —NRC(O)—, —C(O)NR—, —N(R)SO₂—, —SO₂N(R)—, —S—,        —S(O)—, —SO₂—, —OC(O)—, or —C(O)O—; and Y is hydrogen or C₁₋₆        aliphatic optionally substituted with oxo, halogen, NO₂, or CN;        or    -   (l) L is a covalent bond and Y is selected from:        -   (i) C₁₋₆ alkyl substituted with oxo, halogen, NO₂, or CN;        -   (ii) C₂₋₆ alkenyl optionally substituted with oxo, halogen,            NO₂, or CN; or        -   (iii) C₂₋₆ alkynyl optionally substituted with oxo, halogen,            NO₂, or CN; or        -   (iv) a saturated 3-4 membered heterocyclic ring having 1            heteroatom selected from oxygen or nitrogen wherein said            ring is substituted with 1-2 R^(e) groups, wherein each            R^(e) is as defined above and described herein; or        -   (v) a saturated 5-6 membered heterocyclic ring having 1-2            heteroatom selected from oxygen or nitrogen wherein said            ring is substituted with 1-4 R^(e) groups, wherein each            R^(e) is as defined above and described herein; or        -   (vi)

-   -   -    wherein each R, Q, Z, and R^(e) is as defined above and            described herein; or        -   (vii) a saturated 3-6 membered carbocyclic ring, wherein            said ring is substituted with 1-4 R^(e) groups, wherein each            R^(e) is as defined above and described herein; or        -   (viii) a partially unsaturated 3-6 membered monocyclic ring            having 0-3 heteroatoms independently selected from nitrogen,            oxygen, or sulfur, wherein said ring is substituted with 1-4            R^(e) groups, wherein each R^(e) is as defined above and            described herein; or        -   (ix) a partially unsaturated 3-6 membered carbocyclic ring,            wherein said ring is substituted with 1-4 R^(e) groups,            wherein each R^(e) is as defined above and described herein;            or        -   (x)

-   -   -    wherein each R^(e) is as defined above and described            herein; or        -   (xi) a partially unsaturated 4-6 membered heterocyclic ring            having 1-2 heteroatoms independently selected from nitrogen,            oxygen, or sulfur, wherein said ring is substituted with 1-4            R^(e) groups, wherein each R^(e) is as defined above and            described herein; or        -   (xii)

-   -   -    wherein each R and R^(e) is as defined above and described            herein; or        -   (xiii) a 6-membered aromatic ring having 0-2 nitrogens            wherein said ring is substituted with 1-4 R^(e) groups,            wherein each R^(e) group is as defined above and described            herein; or        -   (xiv)

-   -   -    wherein each R^(e) is as defined above and described            herein; or        -   (xv) a 5-membered heteroaryl ring having 1-3 heteroatoms            independently selected from nitrogen, oxygen, or sulfur,            wherein said ring is substituted with 1-3 R^(e) groups,            wherein each R^(e) group is as defined above and described            herein; or        -   (xvi)

-   -   -    wherein each R and R^(e) is as defined above and described            herein; or        -   (xvii) an 8-10 membered bicyclic, saturated, partially            unsaturated, or aryl ring having 0-3 heteroatoms            independently selected from nitrogen, oxygen, or sulfur,            wherein said ring is substituted with 1-4 R^(e) groups,            wherein R^(e) is as defined above and described herein;

    -   (m) L is —C(O)— and Y is selected from:        -   (i) C₁₋₆ alkyl substituted with oxo, halogen, NO₂, or CN; or        -   (ii) C₂₋₆ alkenyl optionally substituted with oxo, halogen,            NO₂, or CN; or        -   (iii) C₂₋₆ alkynyl optionally substituted with oxo, halogen,            NO₂, or CN; or        -   (iv) a saturated 3-4 membered heterocyclic ring having 1            heteroatom selected from oxygen or nitrogen wherein said            ring is substituted with 1-2 R^(e) groups, wherein each            R^(e) is as defined above and described herein; or        -   (v) a saturated 5-6 membered heterocyclic ring having 1-2            heteroatom selected from oxygen or nitrogen wherein said            ring is substituted with 1-4 R^(e) groups, wherein each            R^(e) is as defined above and described herein; or        -   (vi)

-   -   -    wherein each R, Q, Z, and R^(e) is as defined above and            described herein; or        -   (vii) a saturated 3-6 membered carbocyclic ring, wherein            said ring is substituted with 1-4 R^(e) groups, wherein each            R^(e) is as defined above and described herein; or        -   (viii) a partially unsaturated 3-6 membered monocyclic ring            having 0-3 heteroatoms independently selected from nitrogen,            oxygen, or sulfur, wherein said ring is substituted with 1-4            R^(e) groups, wherein each R^(e) is as defined above and            described herein; or

    -   (ix) a partially unsaturated 3-6 membered carbocyclic ring,        wherein said ring is substituted with 1-4 R^(e) groups, wherein        each R^(e) is as defined above and described herein; or        -   (x)

-   -   -    PS wherein each R^(e) is as defined above and described            herein; or        -   (xi) a partially unsaturated 4-6 membered heterocyclic ring            having 1-2 heteroatoms independently selected from nitrogen,            oxygen, or sulfur, wherein said ring is substituted with 1-4            R^(e) groups, wherein each R^(e) is as defined above and            described herein; or        -   (xii)

-   -   -    wherein each R and R^(e) is as defined above and described            herein; or        -   (xiii) a 6-membered aromatic ring having 0-2 nitrogens            wherein said ring is substituted with 1-4 R^(e) groups,            wherein each R^(e) group is as defined above and described            herein; or        -   (xiv)

-   -   -    wherein each R^(e) is as defined above and described            herein; or        -   (xv) a 5-membered heteroaryl ring having 1-3 heteroatoms            independently selected from nitrogen, oxygen, or sulfur,            wherein said ring is substituted with 1-3 R^(e) groups,            wherein each R^(e) group is as defined above and described            herein; or        -   (xvi)

-   -   -    wherein each R and R^(e) is as defined above and described            herein; or        -   (xvii) an 8-10 membered bicyclic, saturated, partially            unsaturated, or aryl ring having 0-3 heteroatoms            independently selected from nitrogen, oxygen, or sulfur,            wherein said ring is substituted with 1-4 R^(e) groups,            wherein R^(e) is as defined above and described herein;

    -   (n) L is —N(R)C(O)— and Y is selected from:        -   (i) C₁₋₆ alkyl substituted with oxo, halogen, NO₂, or CN; or        -   (ii) C₂₋₆ alkenyl optionally substituted with oxo, halogen,            NO₂, or CN; or        -   (iii) C₂₋₆ alkynyl optionally substituted with oxo, halogen,            NO₂, or CN; or        -   (iv) a saturated 3-4 membered heterocyclic ring having 1            heteroatom selected from oxygen or nitrogen wherein said            ring is substituted with 1-2 R^(e) groups, wherein each            R^(e) is as defined above and described herein; or        -   (v) a saturated 5-6 membered heterocyclic ring having 1-2            heteroatom selected from oxygen or nitrogen wherein said            ring is substituted with 1-4 R^(e) groups, wherein each            R^(e) is as defined above and described herein; or        -   (vi)

-   -   -    wherein each R, Q, Z, and R^(e) is as defined above and            described herein; or        -   (vii) a saturated 3-6 membered carbocyclic ring, wherein            said ring is substituted with 1-4 R^(e) groups, wherein each            R^(e) is as defined above and described herein; or        -   (viii) a partially unsaturated 3-6 membered monocyclic ring            having 0-3 heteroatoms independently selected from nitrogen,            oxygen, or sulfur, wherein said ring is substituted with 1-4            R^(e) groups, wherein each R^(e) is as defined above and            described herein; or        -   (ix) a partially unsaturated 3-6 membered carbocyclic ring,            wherein said ring is substituted with 1-4 R^(e) groups,            wherein each R^(e) is as defined above and described herein;            or        -   (x)

-   -   -    wherein each R^(e) is as defined above and described            herein; or        -   (xi) a partially unsaturated 4-6 membered heterocyclic ring            having 1-2 heteroatoms independently selected from nitrogen,            oxygen, or sulfur, wherein said ring is substituted with 1-4            R^(e) groups, wherein each R^(e) is as defined above and            described herein; or        -   (xii)

-   -   -    wherein each R and R^(e) is as defined above and described            herein; or        -   (xiii) a 6-membered aromatic ring having 0-2 nitrogens            wherein said ring is substituted with 1-4 R^(e) groups,            wherein each R^(e) group is as defined above and described            herein; or        -   (xiv)

-   -   -    wherein each R^(e) is as defined above and described            herein; or        -   (xv) a 5-membered heteroaryl ring having 1-3 heteroatoms            independently selected from nitrogen, oxygen, or sulfur,            wherein said ring is substituted with 1-3 R^(e) groups,            wherein each R^(e) group is as defined above and described            herein; or        -   (xvi)

-   -   -    wherein each R and R^(e) is as defined above and described            herein; or        -   (xvii) an 8-10 membered bicyclic, saturated, partially            unsaturated, or aryl ring having 0-3 heteroatoms            independently selected from nitrogen, oxygen, or sulfur,            wherein said ring is substituted with 1-4 R^(e) groups,            wherein R^(e) is as defined above and described herein;

    -   (o) L is a bivalent C₁₋₈ saturated or unsaturated, straight or        branched, hydrocarbon chain; and        -   Y is selected from:        -   (i) C₁₋₆ alkyl substituted with oxo, halogen, NO₂, or CN;        -   (ii) C₂₋₆ alkenyl optionally substituted with oxo, halogen,            NO₂, or CN; or        -   (iii) C₂₋₆ alkynyl optionally substituted with oxo, halogen,            NO₂, or CN; or        -   (iv) a saturated 3-4 membered heterocyclic ring having 1            heteroatom selected from oxygen or nitrogen wherein said            ring is substituted with 1-2 R^(e) groups, wherein each            R^(e) is as defined above and described herein; or        -   (v) a saturated 5-6 membered heterocyclic ring having 1-2            heteroatom selected from oxygen or nitrogen wherein said            ring is substituted with 1-4 R^(e) groups, wherein each            R^(e) is as defined above and described herein; or        -   (vi)

-   -   -    wherein each R, Q, Z, and R^(e) is as defined above and            described herein; or        -   (vii) a saturated 3-6 membered carbocyclic ring, wherein            said ring is substituted with 1-4 R^(e) groups, wherein each            R^(e) is as defined above and described herein; or        -   (viii) a partially unsaturated 3-6 membered monocyclic ring            having 0-3 heteroatoms independently selected from nitrogen,            oxygen, or sulfur, wherein said ring is substituted with 1-4            R^(e) groups, wherein each R^(e) is as defined above and            described herein; or        -   (ix) a partially unsaturated 3-6 membered carbocyclic ring,            wherein said ring is substituted with 1-4 R^(e) groups,            wherein each R^(e) is as defined above and described herein;            or        -   (x)

-   -   -    wherein each R^(e) is as defined above and described            herein; or        -   (xi) a partially unsaturated 4-6 membered heterocyclic ring            having 1-2 heteroatoms independently selected from nitrogen,            oxygen, or sulfur, wherein said ring is substituted with 1-4            R^(e) groups, wherein each R^(e) is as defined above and            described herein; or        -   (xii)

-   -   -    wherein each R and R^(e) is as defined above and described            herein; or        -   (xiii) a 6-membered aromatic ring having 0-2 nitrogens            wherein said ring is substituted with 1-4 R^(e) groups,            wherein each R^(e) group is as defined above and described            herein; or        -   (xiv)

-   -   -    wherein each R^(e) is as defined above and described            herein; or        -   (xv) a 5-membered heteroaryl ring having 1-3 heteroatoms            independently selected from nitrogen, oxygen, or sulfur,            wherein said ring is substituted with 1-3 R^(e) groups,            wherein each R^(e) group is as defined above and described            herein; or        -   (xvi)

-   -   -    wherein each R and R^(e) is as defined above and described            herein; or        -   (xvii) an 8-10 membered bicyclic, saturated, partially            unsaturated, or aryl ring having 0-3 heteroatoms            independently selected from nitrogen, oxygen, or sulfur,            wherein said ring is substituted with 1-4 R^(e) groups,            wherein R^(e) is as defined above and described herein;

    -   (p) L is a covalent bond, —CH₂—, —NH—, —C(O)—, —CH₂NH—, —NHCH₂—,        —NHC(O)—, —NHC(O)CH₂OC(O)—, —CH₂NHC(O)—, —NHSO₂—, —NHSO₂CH₂—,        —NHC(O)CH₂OC(O)—, or —SO₂NH—; and Y is selected from:        -   (i) C₁₋₆ alkyl substituted with oxo, halogen, NO₂, or CN; or        -   (ii) C₂₋₆ alkenyl optionally substituted with oxo, halogen,            NO₂, or CN; or        -   (iii) C₂₋₆ alkynyl optionally substituted with oxo, halogen,            NO₂, or CN; or        -   (iv) a saturated 3-4 membered heterocyclic ring having 1            heteroatom selected from oxygen or nitrogen wherein said            ring is substituted with 1-2 R^(e) groups, wherein each            R^(e) is as defined above and described herein; or        -   (v) a saturated 5-6 membered heterocyclic ring having 1-2            heteroatom selected from oxygen or nitrogen wherein said            ring is substituted with 1-4 R^(e) groups, wherein each            R^(e) is as defined above and described herein; or        -   (vi)

-   -   -    wherein each R, Q, Z, and R^(e) is as defined above and            described herein; or        -   (vii) a saturated 3-6 membered carbocyclic ring, wherein            said ring is substituted with 1-4 R^(e) groups, wherein each            R^(e) is as defined above and described herein; or        -   (viii) a partially unsaturated 3-6 membered monocyclic ring            having 0-3 heteroatoms independently selected from nitrogen,            oxygen, or sulfur, wherein said ring is substituted with 1-4            R^(e) groups, wherein each R^(e) is as defined above and            described herein; or        -   (ix) a partially unsaturated 3-6 membered carbocyclic ring,            wherein said ring is substituted with 1-4 R^(e) groups,            wherein each R^(e) is as defined above and described herein;            or        -   (x)

-   -   -    wherein each R^(e) is as defined above and described            herein; or        -   (xi) a partially unsaturated 4-6 membered heterocyclic ring            having 1-2 heteroatoms independently selected from nitrogen,            oxygen, or sulfur, wherein said ring is substituted with 1-4            R^(e) groups, wherein each R^(e) is as defined above and            described herein; or        -   (xii)

-   -   -    wherein each R and R^(e) is as defined above and described            herein; or        -   (xiii) a 6-membered aromatic ring having 0-2 nitrogens            wherein said ring is substituted with 1-4 R^(e) groups,            wherein each R^(e) group is as defined above and described            herein; or        -   (xiv)

-   -   -    wherein each R^(e) is as defined above and described            herein; or        -   (xv) a 5-membered heteroaryl ring having 1-3 heteroatoms            independently selected from nitrogen, oxygen, or sulfur,            wherein said ring is substituted with 1-3 R^(e) groups,            wherein each R^(e) group is as defined above and described            herein; or        -   (xvi)

-   -   -    wherein each R and R^(e) is as defined above and described            herein; or        -   (xvii) an 8-10 membered bicyclic, saturated, partially            unsaturated, or aryl ring having 0-3 heteroatoms            independently selected from nitrogen, oxygen, or sulfur,            wherein said ring is substituted with 1-4 R^(e) groups,            wherein R^(e) is as defined above and described herein.

In certain embodiments, the Y group of formula I is selected from thoseset forth in Table 1, below, wherein each wavy line indicates the pointof attachment to the rest of the molecule.

TABLE 1 Exemplary Y Groups of Formula I:

  a

  b

  c

  d

  e

  f

  g

  h

  i

  j

  k

  l

  m

  n

  o

  p

  q

  r

  s

  t

  u

  v

  w

  x

  y

  z

  aa

  bb

  cc

  dd

  ee

  ff

  gg

  hh

  ii

  jj

  kk

  ll

  mm

  nn

  oo

  pp

  qq

  rr

  ss

  tt

  uu

  vv

  ww

  xx

  yy

  zz

  aaa

  bbb

  ccc

  ddd

  eee

  fff

  ggg

  hhh

  iii

  jjj

  kkk

  lll

  mmm

  nnn

  ooo

  ppp

  qqq

  rrr

  sss

  ttt

  uuu

  vvv

  qqq

  www

  xxx

  yyy

  zzz

  aaaa

  bbbb

  cccc

  dddd

  eeee

  ffff

  gggg

  hhhh

  iiii

  jjjj

  kkkk

  llll

  mmmm

  nnnn

  oooo

  pppp

  qqqq

  rrrr

  ssss

  tttt

  uuuu

  vvvv

  wwww

  xxxx

  yyyy

  zzzz

  aaaaa

  bbbbb

  ccccc

  dddddwherein each R^(e) is independently a suitable leaving group, NO₂, CN,or oxo.

In certain embodiments, the R³ group of formula I is selected from thoseset forth in Table 2, below, wherein each wavy line indicates the pointof attachment to the rest of the molecule.

TABLE 2 Exemplary R³ Groups:

  a

  b

  c

  d

  e

  f

  g

  h

  i

  j

  k

  l

  m

  n

  o

  p

  q

  r

  s

  t

  u

  v

  w

  x

  y

  z

  aa

  bb

  cc

  dd

  ee

  ff

  gg

  hh

  ii

  jj

  kk

  ll

  mm

  nn

  oo

  pp

  qq

  rr

  ss

  tt

  uu

  vv

  ww

  xx

  yy

  zz

  aaa

  bbb

  ccc

  ddd

  eee

  fff

  ggg

  hhh

  iii

  jjj

  kkk

  lll

  mmm

  nnn

  ooo

  ppp

  qqq

  rrr

  sss

  ttt

  uuu

  vvv

  www

  xxx

  yyy

  zzz

  aaaa

  bbbb

  cccc

  dddd

  eeee

  ffff

  gggg

  hhhh

  iiii

  jjjj

  kkkk

  llll

  mmmm

  nnnn

  oooo

  pppp

  qqqq

  rrrr

  ssss

  tttt

  uuuu

  vvvv

  wwww

  xxxx

  yyyy

  zzzz

  aaaaa

  bbbbb

  ccccc

  ddddd

  eeeee

  fffff

  ggggg

  hhhhh

  iiiii

  jjjjj

  kkkkk

  lllll

  mmmmm

  nnnnn

  ooooo

  ppppp

  qqqqq

  rrrrr

  sssss

  ttttt

  uuuuu

  vvvvv

  wwwww

  xxxxx

  yyyyy

  zzzzz

  aaaaaa

  bbbbbb

  cccccc

  dddddd

  eeeeee

  ffffff

  gggggg

  hhhhhh

  iiiiii

  jjjjjj

  kkkkkk

  llllll

  mmmmmm

  nnnnnn

  oooooo

  pppppp

  qqqqqq

  rrrrrr

  ssssss

  tttttt

  uuuuuu

  vvvvvv

  wwwwww

  xxxxxx

  yyyyyy

  zzzzzz

  a1

  b1

  c1

  d1

  e1

  f1

  g1

  h1

  j1

  k1

  m1

  n1

  o1

  p1

  q1

  r1

wherein each R^(e) is independently a suitable leaving group, NO₂, CN,or oxo.

In certain embodiments, the R′ and R^(1′) groups of formula I areindependently hydrogen or optionally substituted C₁₋₆ aliphatic. In someembodiments, R′ is hydrogen and R^(1′) is C₁₋₄ aliphatic. In otherembodiments, R¹ is hydrogen and R^(1′) is n-propyl.

In certain embodiments, the R′ and R^(1′) groups of formula I are takentogether to form an optionally substituted 3-7 membered carbocyclicring. In some embodiments, the R′ and R^(1′) groups of formula I aretaken together to form an optionally substituted cyclopropyl ring. Insome embodiments, the R′ and R^(1′) groups of formula I are takentogether to form a cyclopropyl ring substituted with ethyl or vinyl.

In some embodiments, R⁴ is H, —NHC(O)R⁵, —NHC(O)OR⁶,

or or R⁴ and R^(z) are taken together with their intervening atoms toform an optionally substituted, saturated or unsaturated 16-22 memberedring having 2-6 heteroatoms independently selected from nitrogen,oxygen, or sulfur.

In certain embodiments, the R⁴ group of formula I is —NHC(O)R⁵. In someembodiments, the R⁴ group of formula I is —NHC(O)OR⁶. In otherembodiments, the R⁴ group of formula I is

In certain embodiments, the R⁴ group of formula I is hydrogen.

In some embodiments, when R⁴ is —NHC(O)R⁵, R⁵ is C₁₋₆ aliphatic or anoptionally substituted group selected from a bridged bicyclic, 6-10membered aryl, 5-10 membered heteroaryl having 1-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, or 4-7 memberedheterocyclyl having 1-2 heteroatoms independently selected fromnitrogen, oxygen, or sulfur.

In some embodiments, when R⁴ is —NHC(O)OR⁶, R⁶ is C₁₋₆ aliphatic or anoptionally substituted group selected from a bridged bicyclic, 6-10membered aryl, 5-10 membered heteroaryl having 1-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, or 4-7 memberedheterocyclyl having 1-2 heteroatoms independently selected fromnitrogen, oxygen, or sulfur.

In some embodiments, the R⁴ group of formula I is an amino acidside-chain group. In some embodiments, the R⁴ group of formula I is anunnatural amino acid side-chain group. In some embodiments, the R⁴ groupof formula I is an aliphatic unnatural amino acid side-chain group. Insome embodiments, the R⁴ group of formula I is an unnatural amino acidside-chain group of alanine substituted with one, two, or threeR^(◯)groups, wherein each R^(◯) is as defined above. In someembodiments, the R⁴ group of formula I is an unnatural amino acidside-chain group of threonine substituted with one, two, or threeR^(◯)groups, wherein each R^(◯) is as defined above. In someembodiments, R^(◯) is methyl.

In some embodiments, the R⁴ group of formula I is a natural amino acidside-chain group.

In certain embodiments, the R⁴ group of formula I is the natural aminoacid side-chain group of alanine (i.e., R⁴ is methyl). In someembodiments, the R⁴ group of formula I is the natural amino acidside-chain group of D-alanine. In some embodiments, the R⁴ group offormula I is the natural amino acid side-chain group of L-alanine.

In other embodiments, the R⁴ group of formula I is the natural aminoacid side-chain group of valine. In some embodiments, the R⁴ group offormula I is the natural amino acid side-chain group of D-valine. Insome embodiments, the R⁴ group of formula I is the natural amino acidside-chain group of L-valine.

In some embodiments, the R⁴ group of formula I consists of a mixture ofamino acid side-chain groups in both the D- and L-configuration. Such R⁴groups are referred to herein as “D,L-mixed amino acid side-chaingroups.” In some embodiments, the ratio of D- to L-amino acid side-chaingroups is selected from any of 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3,1:4; 1:5, and 1:6. Thus, in certain embodiments, the R⁴ group of formulaI is a D,L-mixed alanine side-chain group. In other embodiments, the R⁴group of formula I is a D,L-mixed valine side-chain group.

While not wishing to be bound by any particular theory, it is believedthat for compounds of formula I, having an amino acid side-chain groupin the D-configuration is useful in allowing a compound to adopt anorientation conducive to binding HCV protease.

In certain embodiments, the R⁵ and R⁷ groups of formula I areindependently optionally substituted groups selected from optionallysubstituted group selected from C₁₋₆ aliphatic, 6-10 membered aryl, 5-10membered heteroaryl having 1-4 heteroatoms independently selected fromnitrogen, oxygen, or sulfur, or 4-7 membered heterocyclyl having 1-2heteroatoms independently selected from nitrogen, oxygen, or sulfur. Insome embodiments, R⁵ is an optionally substituted 5-10 memberedheteroaryl having 1-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur, and R⁷ is an optionally substituted C₁₋₆ aliphaticgroup. In some embodiments, R⁵ is

and R⁷ is cyclohexyl.

In certain embodiments, R⁴ is —NHC(O)R⁵, wherein R⁵ is independently—N(R)₂ or an optionally substituted group selected from C₁₋₆ aliphatic.In some embodiments, R⁵ is —N(R)₂ and each R is independently hydrogen,optionally substituted C₁₋₆ aliphatic, or two R on the same nitrogenatom are taken together with the nitrogen to form a 4-7 memberedheterocyclic ring having 1-2 heteroatoms independently selected fromnitrogen, oxygen, or sulfur. In some embodiments, R⁵ is —N(R)₂ and eachR is independently hydrogen or t-butyl.

In certain embodiments, the R⁵ group of formula I is an optionallysubstituted 5-10 membered heteroaryl ring having 1-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, R⁵ is an optionally substituted 6 membered heteroaryl ringhaving 1-2 nitrogens. In certain embodiments, R⁵ is piperazinyl.

In certain embodiments, the R⁷ group of formula I is an optionallysubstituted C₁₋₆ aliphatic group. In some embodiments, R⁷ is a branchedC₁₋₅ alkyl group. In other embodiments, R⁷ is cyclopentyl or cyclohexyl.

In certain embodiments, R^(z) is

In certain embodiments, R^(z) is

In certain embodiments, R^(z) is

In certain embodiments, R^(z) is

In certain embodiments, R^(z) is

In certain embodiments, R^(z) is

In certain embodiments, R^(z) is

In certain embodiments, R^(z) is

In certain embodiments, R^(z) is

In some embodiments, R^(y) is halogen. In other embodiments, R^(y) isC₁₋₄ aliphatic. In certain embodiments, R^(y) is fluoro. In certainembodiments, R^(y) is chloro. In certain embodiments, R^(y) is bromo. Incertain embodiments, R^(y) is iodo. In other embodiments, R^(y) isvinyl.

In some embodiments, m is an integer between 1 and 3, inclusive. In someembodiments, m is 1. In some embodiments, m is 2. In some embodiments, mis 3.

In some embodiments, s is an integer between 1 and 3, inclusive. In someembodiments, s is 0. In some embodiments, s is 1. In some embodiments, sis 2. In some embodiments, s is 3. In some embodiments, s is 4.

In some embodiments, t is an integer between 1 and 3, inclusive. In someembodiments, t is 0. In some embodiments, t is 1. In some embodiments, tis 2. In some embodiments, t is 3. In some embodiments, t is 4.

In certain embodiments, the R^(2a) group of formula I is —OH. In otherembodiments, the R^(2a) group of formula I is —NHSO₂R², wherein R² is asdefined above and described herein. Thus, the present invention providesa compound of formula I-a or I-b:

or a pharmaceutically acceptable salt thereof, wherein each of R¹,R^(1′), R², R³, R⁴, and R^(z) is as defined above for formula I anddescribed in classes and subclasses above and herein.

In certain embodiments, the R² group of formula I-b is —N(R)₂. In otherembodiments, the R² group of formula I-b is an optionally substitutedgroup selected from C₃₋₇ cycloalkyl, 6-10 membered aryl, 5-10 memberedheteroaryl having 1-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur, or 4-7 membered heterocyclyl having 1-2 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In certainembodiments, R² is C₃₋₇ cycloalkyl or 6-10 membered aryl. In someembodiments, R² is optionally substituted 6-10 membered aryl. In someembodiments, R² is phenyl. In certain embodiments, R² is cyclopropyl.

In certain embodiments, R² is selected from C₃₋₇ cycloalkyl, a bridgedbicyclic, 6-10 membered aryl, 5-10 membered heteroaryl having 1-4heteroatoms independently selected from nitrogen, oxygen, or sulfur, or4-7 membered heterocyclyl having 1-2 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur.

In certain embodiments, the R³ group of formula I is a warhead group. Insome embodiments, the R³ and R′ groups of formula I are taken togetherwith their intervening atoms to form an optionally substituted saturatedor unsaturated 12-18 membered ring having 2-6 heteroatoms independentlyselected from nitrogen oxygen, or sulfur, wherein the ring formedthereby comprises a warhead group. In some embodiments, R³ and a ringformed by R¹ and R^(1′) are taken together with their intervening atomsto form an optionally substituted saturated or unsaturated 12-18membered ring having 2-6 heteroatoms independently selected fromnitrogen oxygen, or sulfur, wherein the ring formed thereby comprises awarhead group.

As defined generally above, the ring formed by the R³ and R¹ groups offormula I comprises a warhead group. As used herein, the phrase“comprises a warhead group” means that the ring formed by R³ and R¹ iseither substituted with a warhead group or has such a warhead groupincorporated within the ring. For example, the ring formed by R³ and R¹may be substituted with an -L-Y warhead group, wherein such groups areas described herein. Alternatively, the ring formed by R³ and R¹ has theappropriate features of a warhead group incorporated within the ring.For example, the ring formed by R³ and R¹ may include one or more unitsof unsaturation and optional substituents and/or heteroatoms which, incombination, result in a moiety that is capable of covalently modifyingHCV protease in accordance with the present invention. In certainembodiments, the ring formed by R³ and R¹ is optionally substituted atthe α-, β-, or γ-position with respect to the carbon to which R⁴ isattached.

It will be appreciated that when R³ and R¹ are taken together with theirintervening atoms to form an optionally substituted saturated orunsaturated 12-18 membered ring having 2-6 heteroatoms independentlyselected from nitrogen oxygen, or sulfur, such compounds include thosewherein R³ and a ring formed by R¹ and R^(1′) are taken together.

Exemplary compounds of formula I wherein R³ and a ring formed by R¹ andR^(1′) are taken together include those of formula I-c-1, I-c-2, I-c-3,I-c-4, I-c-5-, and I-c-6:

or a pharmaceutically acceptable salt thereof, wherein each of R^(2a),R⁴, and R^(z) is as defined above and described in classes andsubclasses herein. It will be appreciated that, although formulae I-c-1,I-c-2, I-c-3, I-c-4, I-c-5, and I-c-6 depict a cyclopropyl ring formedby R′ and R″, this group is depicted for the purposes of exemplificationand therefore other R′ and R^(1′) groups, as described herein, arecontemplated.

Exemplary such compounds include those set forth in Table 3, infra.

While compounds of formulae I-c-1, I-c-2, I-c-3, I-c-4, I-c-5, and I-c-6are depicted as having (Z)-double bond stereochemistry in themacrocyclic ring, it will be understood that, in certain embodiments,compounds of formulae I-c-1, I-c-2, I-c-3, I-c-4, I-c-5, and I-c-6 maybe provided having (E)-double bond stereochemistry in the macrocylicring. In some embodiments, mixtures of both stereoisomers are provided.In other embodiments, compounds of formulae I-c-1, I-c-2, I-c-3, I-c-4,I-c-5, and I-c-6 may be treated under suitable conditions to saturatethe double bond.

In certain embodiments, R¹ and R^(1′) are taken together to form anoptionally substituted 3-7 membered carbocyclic ring. In someembodiments, such compounds are of formula I-d:

or a pharmaceutically acceptable salt thereof, wherein each R^(2a), R³,R⁴, R^(z), R^(◯) is as defined in formula I and described in classes andsubclasses above and herein.

In some embodiments, R^(◯) is an optionally substituted group selectedfrom C₁₋₆ aliphatic. In some embodiments, R^(◯) is ethyl. In otherembodiments, R^(◯) is vinyl.

Exemplary R³ groups of formula I-d include those described above andherein, as well as those depicted in Table 3, below.

In certain embodiments, R⁴ and R^(z) are taken together with theirintervening atoms to form an optionally substituted, saturated orunsaturated 16-22 membered ring having 2-6 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur. In some embodiments, R⁴ andR^(z) are taken together with their intervening atoms to form anoptionally substituted, unsaturated 18-22 membered ring having 3-5heteroatoms independently selected from nitrogen, oxygen, or sulfur. Insome embodiments, the ring formed by R⁴ and R^(z) is substituted withone or more R^(m) groups, wherein each occurrence of R^(m) isindependently halogen, —OR^(◯); —CN; —SCN; —SR^(◯); —SOR^(◯); —SO₂R^(◯);—NO₂; —N(R^(◯))₂; —NHC(O)R^(◯), or an optionally substituted groupselected from the group consisting of C₁₋₆ aliphatic and C₃₋₇cycloalkyl. In certain embodiments, the present invention providescompounds of formula I-e or I-f:

or a pharmaceutically acceptable salt thereof, wherein each of m, s, t,R^(2a), R³, R^(y), and R^(◯) is as defined in formula I and described inclasses and subclasses above and herein;p is an integer from 1 to 6, inclusive; andeach occurrence of R^(m) is independently halogen, —OR^(◯); —CN;—N(R^(◯))₂; or an optionally substituted group selected from the groupconsisting of C₁₋₆ aliphatic and C₃₋₇ cycloalkyl.

In some embodiments, p is 1. In some embodiments, p is 2.

In certain embodiments, R^(m) is C₁₋₆ aliphatic. In some embodiments,R^(m) is methyl.

In some embodiments, R^(◯) is an optionally substituted group selectedfrom C₁₋₆ aliphatic. In some embodiments, R^(◯) is ethyl. In otherembodiments, R^(◯) is vinyl.

Exemplary R³ groups of formulae I-e and I-f include those describedherein and depicted in Table 3, below.

While compounds of formulae I-e and I-f are depicted as having either(Z) or (E) double bond stereochemistry in the macrocyclic ring, it willbe understood that, in certain embodiments, compounds of formulae I-eand I-f may be provided having (E)-double bond stereochemistry in themacrocyclic ring. In certain embodiments, compounds of formulae I-e andI-f may be provided having (Z)-double bond stereochemistry in themacrocylic ring. In some embodiments, mixtures of both stereoisomers areprovided. In other embodiments, compounds of formulae I-e and I-f may betreated under suitable conditions to saturate the double bond, therebyforming a compound of formula I-g or I-h:

or a pharmaceutically acceptable salt thereof.

In certain embodiments, R⁴ and R^(z) are taken together as describedabove, and R³ and a ring formed by R¹ and R^(1′) are taken together asdescribed above, to form novel bimacrocyclic compounds. In certainembodiments, the ring formed by R⁴ and R^(z) is substituted with one ormore R^(m) groups as described above for formulae I-e and I-f. In someembodiments, the macrocyclic ring formed by R³ and a ring formed by R¹and R^(1′) is substituted with an -L-Y warhead group to provide acompound of formula I-j or I-k:

or a pharmaceutically acceptable salt thereof; wherein each

independently represents a single or double bond. Methods of preparingsuch compounds, in addition to those described herein for the synthesisof other macrocycles and compounds incorporating a warhead, includethose described by McCauley, J. A. et al., Angew. Chem. Int. Ed., 2008,47, pp. 9104-7.

In some embodiments, a methylene unit of the macrocyclic ring formed byR³ and a ring formed by R¹ and R^(1′) is replaced by an L-Y moiety toprovide a compound of formula I-m or I-n:

or a pharmaceutically acceptable salt thereof; wherein each

independently represents a single or double bond.

As described above and herein, in certain embodiments, the R⁴ group forcompounds of formula I is hydrogen. In certain embodiments, the presentinvention provides a compound of formula II-a or II-b:

wherein each of the R¹, R^(1′), R², R³, and R^(z) groups is as definedfor formula I above and described in classes and subclasses herein.

Exemplary compounds of formula I are set forth in Table 3 below.

TABLE 3 Exemplary Compounds of Formula I

  I-1 

  I-2 

  I-3 

  I-4 

  I-5 

  I-6 

  I-7 

  I-8 

  I-9 

  I-10

  I-11

  I-12

  I-13

  I-14

  I-15

  I-16

  I-17

  I-18

  I-19

  I-20

  I-21

  I-22

  I-23

  I-24

  I-25

  I-26

  I-27

  I-28

  I-29

  I-30

  I-31

  I-32

  I-33

  I-34

  I-35

  I-36

  I-37

  I-38

  I-39

  I-40

  I-41

  I-42

  I-43

  I-44

  I-45

  I-46

  I-47

  I-48

  I-49

  I-50

  I-51

  I-52

  I-53

  I-54

  I-55

  I-56

  I-57

  I-58

  I-59

  I-60

  I-61

  I-62

  I-63

  I-64

  I-65

  I-66

  I-67

  I-68

  I-69

  I-70

  I-71

  I-72

  I-73

  I-74

  I-75

  I-76

  I-77

  I-78

  I-79

In certain embodiments, the present invention provides any compounddepicted in Table 3, above, or a pharmaceutically acceptable saltthereof.

As defined generally above, R³ is a warhead group. Without wishing to bebound by any particular theory, it is believed that such R³ groups, i.e.warhead groups, are particularly suitable for covalently binding to akey cysteine residue in the binding domain of HCV protease. One ofordinary skill in the art will appreciate that HCV protease, and mutantsthereof, have a cysteine residue in the binding domain. In certainembodiments, compounds of the present invention have a warhead groupcharacterized in that inventive compounds may target the C159 cysteineresidue of HCV protease.

Thus, in some embodiments, R³ is characterized in that the -L-Y moietyis capable of covalently binding to a cysteine residue therebyirreversibly inhibiting the enzyme. In certain embodiments, the cysteineresidue is Cys159 of HCV protease, or a mutant thereof, where theprovided residue numbering is in accordance with Uniprot (code Q91RS4).

One of ordinary skill in the art will recognize that a variety ofwarhead groups, as defined herein, are suitable for such covalentbonding. Such R³ groups include, but are not limited to, those describedherein and depicted in Table 3, supra. This phenomenon may be determinedby performing mass spectroscopic experiments using the protocoldescribed in detail in Examples 17 through 21, infra.

According to another aspect, the present invention provides a conjugatecomprising HCV protease, or a mutant thereof, covalently bonded to aninhibitor at Cys159. In some embodiments, the inhibitor is covalentlybonded via a linker moiety.

In certain embodiments, the present invention provides a conjugate ofthe formula Cys159-linker-inhibitor moiety. One of ordinary skill in theart will recognize that the “linker” group corresponds to an -L-Ywarhead group as described herein. Accordingly, in certain embodiments,the linker group is as defined for -L-Y was defined above and describedin classes and subclasses herein. It will be appreciated, however, thatthe linker group is bivalent and, therefore, the corresponding -L-Ygroup is also intended to be bivalent resulting from the reaction of thewarhead with the Cys 159 of HCV protease, or a mutant thereof.

In certain embodiments, the inhibitor moiety is a compound of formula A:

wherein each of the R¹, R^(1′), R^(2a), R⁴, and R^(z) groups of formulaA is as defined for formula I above and described in classes andsubclasses herein. Thus, in certain embodiments, the present inventionprovides a conjugate of the formula:

wherein each of the R¹, R^(1′), R^(2a), R⁴, and R^(z) groups of theconjugate is as defined for formula I above and described in classes andsubclasses herein.

In some embodiments, R³ is characterized in that the -L-Y moiety iscapable of covalently binding to a cysteine residue thereby irreversiblyinhibiting the enzyme. In certain embodiments, the cysteine residue isCys16 of HCV protease, or a mutant thereof, where the provided residuenumbering is in accordance with Uniprot (code Q91RS4).

According to another aspect, the present invention provides a conjugatecomprising HCV protease, or a mutant thereof, covalently bonded to aninhibitor at Cys16. In some embodiments, the inhibitor is covalentlybonded via a linker moiety.

In certain embodiments, the present invention provides a conjugate ofthe formula Cys16-linker-inhibitor moiety. One of ordinary skill in theart will recognize that the “linker” group corresponds to an -L-Ywarhead group as described herein. Accordingly, in certain embodiments,the linker group is as defined for -L-Y was defined above and describedin classes and subclasses herein. It will be appreciated, however, thatthe linker group is bivalent and, therefore, the corresponding -L-Ygroup is also intended to be bivalent resulting from the reaction of thewarhead with the Cys16 of HCV protease, or a mutant thereof.

In certain embodiments, the inhibitor moiety is a compound of formulaA-1:

wherein each of the R¹, R¹, R^(2a), R⁴, and R^(z) groups of formula A-1is as defined for formula I above and described in classes andsubclasses herein. Thus, in certain embodiments, the present inventionprovides a conjugate of the formula:

wherein each of the R¹, R^(1′), R^(2a), R⁴, and R^(z) groups of theconjugate is as defined for formula I above and described in classes andsubclasses herein.

General Methods of Providing the Present Compounds

In certain embodiments, the present compounds are generally preparedaccording to Scheme 1 set forth below:

In one aspect, the present invention provides methods for preparingcompounds of formula I, according to the steps depicted in Scheme 1above wherein each variable is as defined and described herein and eachPG is a suitable protecting group. At step S-1, an N-protected (e.g.Boc) proline derivative of formula A is condensed with analpha-aminoester of formula B using peptide coupling conditions to givea dipeptide of formula C. Suitable peptide coupling conditions are wellknown in the art and include those described in detail in PCTpublication number WO2002094822 (U.S. Pat. No. 6,825,347), the entiretyof which is hereby incorporated by reference. Unless otherwiseindicated, said conditions are referenced as suitable peptide couplingconditions throughout this application.

At step S-2, the ester group is hydrolyzed with a suitable base andsubsequently neutralized to give a dipeptide of formula D. Suitablebases include, but are not limited to, alkaline metals, alkaline earthmetal hydroxides, and combinations thereof. In some embodiments, thebase is lithium hydroxide.

At step S-3, a dipeptide of formula D is coupled with a sulfonamide offormula E using suitable peptide coupling conditions to give anacylsulfonamide of formula F.

At step S-4, cleavage of the protective group (e.g. Boc removal) from adipeptide of formula F gives an amine of formula G. In certainembodiments, cleavage of the Boc group is achieved by contacting acompound of formula F with a mineral or organic acid in a halogenatedhydrocarbon solvent. In some embodiments, In some embodiments, the acidis trifluoroacetic acid and the solvent is dichloromethane.

At step S-5, all amine of formula G is coupled with an carboxylic acidof formula H using suitable peptide coupling conditions to give anintermediate compound of formula I-0.

Intermediate compound of formula I-0 is converted to compounds offormula I in steps which are described as examples herein.

As defined generally above, the PG group of formulae A, C, D, and F is asuitable amino protecting group. Suitable amino protecting groups arewell known in the art and include those described in detail inProtecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts,3^(rd) edition, John Wiley & Sons, 1999, the entirety of which isincorporated herein by reference. Protected amines are well known in theart and include those described in detail in Greene (1999). Suitablemono-protected amines further include, but are not limited to,aralkylamines, carbamates, allyl amines, amides, and the like. Examplesof suitable mono-protected amino moieties includet-butyloxycarbonylamino (—NHBOC), ethyloxycarbonylamino,methyloxycarbonylamino, trichloroethyloxycarbonylamino,allyloxycarbonylamino (—NHAlloc), benzyloxocarbonylamino (—NHCBZ),allylamino, benzylamino (—NHBn), fluorenylmethylcarbonyl (—NHFmoc),formamido, acetamido, chloroacetamido, dichloroacetamido,trichloroacetamido, phenylacetamido, trifluoroacetamido, benzamido,t-butyldiphenylsilyl, and the like. Suitable di-protected amines includeamines that are substituted with two substituents independently selectedfrom those described above as mono-protected amines, and further includecyclic imides, such as phthalimide, maleimide, succinimide, and thelike.

In other embodiments, the present compounds are generally preparedaccording to Scheme 2 set forth below.

In one aspect, the present invention provides methods for preparingcompounds of formula I, according to the steps depicted in Scheme 1above. At step S-6, removal of the Boc group from a dipeptide of formulaC is achieved under acid-catalyzed conditions to give a dipeptide esterof formula J.

At step S-7, a dipeptide ester of formula J is condensed with afunctionalized amino acid of formula H using suitable peptide couplingconditions to give a tripeptide ester of formula K which is furtherconverted to a tripeptide ester of formula L in steps which aredescribed as examples herein.

At step S-8, the ester group on a compound of formula L is hydrolyzedwith a suitable base and subsequently neutralized to give a tripeptideof formula M. Suitable bases include, but are not limited to, alkalinemetals, alkaline earth metal hydroxides, and combinations thereof. Insome embodiments, the base is lithium hydroxide.

At step S-9, a tripeptide of formula M is condensed with a sulfonamideof formula E using suitable peptide coupling conditions to givecompounds of formula I.

The PG group of formulae C, H, and K is a suitable amino protectinggroup as described above.

4. Uses, Formulation and Administration

Pharmaceutically Acceptable Compositions

According to another embodiment, the invention provides a compositioncomprising a compound of this invention or a pharmaceutically acceptablederivative thereof and a pharmaceutically acceptable carrier, adjuvant,or vehicle. The amount of compound in compositions of this invention issuch that is effective to measurably inhibit HCV protease, or a mutantthereof, in a biological sample or in a patient. In certain embodiments,the amount of compound in compositions of this invention is such that iseffective to measurably inhibit HCV protease, or a mutant thereof, in abiological sample or in a patient. In certain embodiments, a compositionof this invention is formulated for administration to a patient in needof such composition. In some embodiments, a composition of thisinvention is formulated for oral administration to a patient.

The term “patient,” as used herein, means an animal, preferably amammal, and most preferably a human.

The term “pharmaceutically acceptable carrier, adjuvant, or vehicle”refers to a non-toxic carrier, adjuvant, or vehicle that does notdestroy the pharmacological activity of the compound with which it isformulated. Pharmaceutically acceptable carriers, adjuvants or vehiclesthat may be used in the compositions of this invention include, but arenot limited to, ion exchangers, alumina, aluminum stearate, lecithin,serum proteins, such as human serum albumin, buffer substances such asphosphates, glycine, sorbic acid, potassium sorbate, partial glyceridemixtures of saturated vegetable fatty acids, water, salts orelectrolytes, such as protamine sulfate, disodium hydrogen phosphate,potassium hydrogen phosphate, sodium chloride, zinc salts, colloidalsilica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-basedsubstances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers,polyethylene glycol and wool fat.

A “pharmaceutically acceptable derivative” means any non-toxic salt,ester, salt of an ester or other derivative of a compound of thisinvention that, upon administration to a recipient, is capable ofproviding, either directly or indirectly, a compound of this inventionor an inhibitorily active metabolite or residue thereof.

As used herein, the term “inhibitorily active metabolite or residuethereof” means that a metabolite or residue thereof is also an inhibitorof HCV protease, or a mutant thereof.

Compositions of the present invention may be administered orally,parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir. The term “parenteral”as used herein includes subcutaneous, intravenous, intramuscular,intra-articular, intra-synovial, intrasternal, intrathecal,intrahepatic, intralesional and intracranial injection or infusiontechniques. Preferably, the compositions are administered orally,intraperitoneally or intravenously. Sterile injectable forms of thecompositions of this invention may be aqueous or oleaginous suspension.These suspensions may be formulated according to techniques known in theart using suitable dispersing or wetting agents and suspending agents.The sterile injectable preparation may also be a sterile injectablesolution or suspension in a non-toxic parenterally acceptable diluent orsolvent, for example as a solution in 1,3-butanediol. Among theacceptable vehicles and solvents that may be employed are water,Ringer's solution and isotonic sodium chloride solution. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium.

For this purpose, any bland fixed oil may be employed includingsynthetic mono- or di-glycerides. Fatty acids, such as oleic acid andits glyceride derivatives are useful in the preparation of injectables,as are natural pharmaceutically-acceptable oils, such as olive oil orcastor oil, especially in their polyoxyethylated versions. These oilsolutions or suspensions may also contain a long-chain alcohol diluentor dispersant, such as carboxymethyl cellulose or similar dispersingagents that are commonly used in the formulation of pharmaceuticallyacceptable dosage forms including emulsions and suspensions. Othercommonly used surfactants, such as Tweens, Spans and other emulsifyingagents or bioavailability enhancers which are commonly used in themanufacture of pharmaceutically acceptable solid, liquid, or otherdosage forms may also be used for the purposes of formulation.

Pharmaceutically acceptable compositions of this invention may be orallyadministered in any orally acceptable dosage form including, but notlimited to, capsules, tablets, aqueous suspensions or solutions. In thecase of tablets for oral use, carriers commonly used include lactose andcorn starch. Lubricating agents, such as magnesium stearate, are alsotypically added. For oral administration in a capsule form, usefuldiluents include lactose and dried cornstarch. When aqueous suspensionsare required for oral use, the active ingredient is combined withemulsifying and suspending agents. If desired, certain sweetening,flavoring or coloring agents may also be added.

Alternatively, pharmaceutically acceptable compositions of thisinvention may be administered in the form of suppositories for rectaladministration. These can be prepared by mixing the agent with asuitable non-irritating excipient that is solid at room temperature butliquid at rectal temperature and therefore will melt in the rectum torelease the drug. Such materials include cocoa butter, beeswax andpolyethylene glycols.

Pharmaceutically acceptable compositions of this invention may also beadministered topically, especially when the target of treatment includesareas or organs readily accessible by topical application, includingdiscases of the eye, the skin, or the lower intestinal tract. Suitabletopical formulations are readily prepared for each of these areas ororgans.

Topical application for the lower intestinal tract can be effected in arectal suppository formulation (see above) or in a suitable enemaformulation. Topically-transdermal patches may also be used.

For topical applications, provided pharmaceutically acceptablecompositions may be formulated in a suitable ointment containing theactive component suspended or dissolved in one or more carriers.Carriers for topical administration of compounds of this inventioninclude, but are not limited to, mineral oil, liquid petrolatum, whitepetrolatum, propylene glycol, polyoxyethylene, polyoxypropylenecompound, emulsifying wax and water. Alternatively, providedpharmaceutically acceptable compositions can be formulated in a suitablelotion or cream containing the active components suspended or dissolvedin one or more pharmaceutically acceptable carriers. Suitable carriersinclude, but are not limited to, mineral oil, sorbitan monostearate,polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol,benzyl alcohol and water.

For ophthalmic use, provided pharmaceutically acceptable compositionsmay be formulated as micronized suspensions in isotonic, pH adjustedsterile saline, or, preferably, as solutions in isotonic, pH adjustedsterile saline, either with or without a preservative such asbenzylalkonium chloride. Alternatively, for ophthalmic uses, thepharmaceutically acceptable compositions may be formulated in anointment such as petrolatum.

Pharmaceutically acceptable compositions of this invention may also beadministered by nasal aerosol or inhalation. Such compositions areprepared according to techniques well-known in the art of pharmaceuticalformulation and may be prepared as solutions in saline, employing benzylalcohol or other suitable preservatives, absorption promoters to enhancebioavailability, fluorocarbons, and/or other conventional solubilizingor dispersing agents.

Most preferably, pharmaceutically acceptable compositions of thisinvention are formulated for oral administration. Such formulations maybe administered with or without food. In some embodiments,pharmaceutically acceptable compositions of this invention areadministered without food. In other embodiments, pharmaceuticallyacceptable compositions of this invention are administered with food.

The amount of compounds of the present invention that may be combinedwith the carrier materials to produce a composition in a single dosageform will vary depending upon the host treated, the particular mode ofadministration. Preferably, provided compositions should be formulatedso that a dosage of between 0.01-100 mg/kg body weight/day of theinhibitor can be administered to a patient receiving these compositions.

It should also be understood that a specific dosage and treatmentregimen for any particular patient will depend upon a variety offactors, including the activity of the specific compound employed, theage, body weight, general health, sex, diet, time of administration,rate of excretion, drug combination, and the judgment of the treatingphysician and the severity of the particular disease being treated. Theamount of a compound of the present invention in the composition willalso depend upon the particular compound in the composition.

Uses of Compounds and Pharmaceutically Acceptable Compositions

Compounds and compositions described herein are generally useful for theinhibition of HCV protease activity and/or the activity of a mutantthereof. Thus, provided compounds are useful for treating non-A, non-Bhepatitis, including hepatitis C.

HCV is an extremely variable virus that forms polymorphic swarms ofvariants within the host. Worldwide, six different genotypes have nowbeen defined (Simmonds et al., Hepatology, Vol. 42, No. 4, 2005). Thesegenotypes have been further classified into more closely related,genetically distinct subtypes. Comparative sequence portions, known asconsensus sequences, are set forth in Table 3a, below. HCV genotypes andsubtypes are distributed differently in different parts of the world,and certain genotypes predominate in certain areas. Genotypes 1-3 arewidely distributed throughout the world. Subtype la is prevalent inNorth and South America, Europe, and Australia. Subtype 1b is common inNorth America and Europe, and is also found in parts of Asia. Genotype 2is present in most developed countries, but is less common than genotype1 (http://www.hcvadvocate.org/hepatitis/factsheets_pdf/genotype_FS.pdf).Other genotypes are prevalent in ex-US patient populations and aretherefore important targets.

Notably, a cysteine located at amino acid position 159 in genotype 1b isconserved in all genotypes and subtypes of HCV NS3 sequenced to date,although the amino acid position may be different in other genotypes andsubtypes. Targeting this cysteine residue with irreversible inhibitorsshould enable the development of agents which are effective againstmultiple HCV genotypes.

As described herein, the present invention provides irreversibleinhibitors of one or more HCV protease genotypes, and variants thereof.Such compounds, comprising a warhead group designated as R³, includethose of formulae I, I-a, I-b, I-c-1, I-c-2, I-c-3, I-c-4, I-c-5, I-c-6,I-d, I-e, I-f, I-g, I-h, II-a, and II-b, as described herein. In someembodiments, R³ is characterized in that the -L-Y moiety is capable ofcovalently binding to a cysteine residue thereby irreversibly inhibitingthe enzyme. Without wishing to be bound by any particular theory, it isbelieved that such R³ groups, i.e. warhead groups, are particularlysuitable for covalently binding to a key cysteine residue in the bindingdomain of one or more HCV protease genotypes or variants thereof. Insome embodiments, one or more genotypes inhibited by compounds of thepresent invention include 1a, 1b, 2a, and 3a. In certain embodiments,one or more such variants include A156T, A156S, D168V, D168A, and R155K.

One of ordinary skill in the art will appreciate that HCV proteasegenotypes and variants thereof have one or more cysteine residues nearthe binding domain. Without wishing to be bound by any particulartheory, it is believed that proximity of a warhead group to the cysteineof interest facilitates covalent modification of that cysteine by thewarhead group. In some embodiments, the cysteine residue of interest isCys159 of HCV protease subtype Ib, or a variant thereof, where theprovided residue numbering is in accordance with Uniprot (code Q91RS4).Cysteine residues of other HCV protease genotypes and subtypes suitablefor covalent modification by irreversible inhibitors of the presentinvention include those summarized in Table 3a, below, where the boldand underlined “C” refers to a cysteine residue conserved at anequivalent position to Cys 159 of HCV protease subtype Ib.

TABLE 3a HCV Representative genotype/ Sequence Sequence subtypePortion^(a) Patient ID Identifier 1a GHAVGLFRAAV C TRGVAKAV_.H77.NC_004102 SEQ ID NO: 1 1a GHAVGIFRAAV C TRGVAKAVCH.BID-V271.EU482858 SEQ ID NO: 2 1a GHAVGIFRAAV C TRGVAKAVDE.BID-V25.EU482831 SEQ ID NO: 3 1a GHAVGLFRAAV C TRGVAKAVUS.H77-H21.AF011753 SEQ ID NO: 4 1b GHAVGIFRAAV C TRGVAKAVAU.HCV-A.AJ000009 SEQ ID NO: 5 1b GHVVGIFRAAV C TRGVAKAVCH.BID-V272.EU482859 SEQ ID NO: 6 1b GHAVGIFRAAV C TRGVAKAVJP.HCV-BK.M58335 SEQ ID NO: 7 1c GHAVGIFRAAV C TRGVAKAV ID.HC-G9.D14853SEQ ID NO: 8 1c GHVAGIFRAAV C TRGVAKAV IN.AY051292.AY051292 SEQ ID NO: 92a GHAVGIFRAAV C SRGVAKSI JP.AY746460.AY746460 SEQ ID NO: 10 2aGHAVGIFRAAV C SRGVAKSI JP.JCH-6.AB047645 SEQ ID NO: 11 2a GHAVGIFRAAV CSRGVAKSI _.G2AK1.AF169003 SEQ ID NO: 12 2b GHAVGLFRAAV C ARGVAKSIJP.HC-J8.D10988 SEQ ID NO: 13 2b GHAVGLFRAAV C ARGVAKSIJP.MD2b1-2.AY232731 SEQ ID NO: 14 2c GHAVGIFRAAV C SRGVAKSI_.BEBE1.D50409 SEQ ID NO: 15 2i AHAVGIFRAAV C SRGVAKSI VN.D54.DQ155561SEQ ID NO: 16 2k GHAVGIFRAAI C TRGAAKSI MD.VAT96.AB031663 SEQ ID NO: 173a GHVAGIFRAAV C TRGVAKAL CH.452.DQ437509 SEQ ID NO: 18 3a GHVAGIFRAAV CTRGVAKAL DE.HCVCENS1.X76918 SEQ ID NO: 19 3a GHVAGIFRAAV C TRGVAKALID.ps23.EU315121 SEQ ID NO: 20 3b GHVMGIFIAVV C TRGVAKALIN.RG416.DQ284965 SEQ ID NO: 21 3b GHVVGIFRAAV C TRGVAKALJP.HCV-Tr.D49374 SEQ ID NO: 22 3k GHVAGIFRAAV C TRGVAKAL ID.JK049.D63821SEQ ID NO: 23 4a GHAAGIFRAAV C TRGVAKAV EG.Eg9.DQ988077 SEQ ID NO: 24 4aGHAAGLFRAAV C TRGVAKAV _.01-09.DQ418782 SEQ ID NO: 25 4a GHAAGLFRAAV CTRGVAKAV _.F753.DQ418787 SEQ ID NO: 26 4d GHAAGIFRAAV C TRGVAKAV_.03-18.DQ418786 SEQ ID NO: 27 4d GHAAGIFRAAV C TRGVAKTV _.24.DQ516083SEQ ID NO: 28 4f GHAVGIFRAAV C TRGVAKAV FR.IFBT84.EF589160 SEQ ID NO: 294f GHAVGIFRAAV C TRGVAKAV FR.IFBT88.EF589161 SEQ ID NO: 30 5aGHVVGVFRAAV C TRGVAKAL GB.EUH1480.Y13184 SEQ ID NO: 31 5a GHVVGVFRAAV CTRGVAKAL ZA.SA13.AF064490 SEQ ID NO: 32 6a GHVVGLFRAAV C TRGVAKSLHK.6a74.DQ480524 SEQ ID NO: 33 6a GHVVGLFRAAV C TRGVAKSLHK.6a77.DQ480512 SEQ ID NO: 34 6a GHVVGLFRAAV C TRGVAKSL HK.EUHK2.Y12083SEQ ID NO: 35 6b GHVVGLFRAAV C TRGVAKAL _.Th580.NC_009827 SEQ ID NO: 366c GHVVGLFRAAV C TRGVAKAL TH.Th846.EF424629 SEQ ID NO: 37 6d DHVVGLFRAAVC TRGVAKAL VN.VN235.D84263 SEQ ID NO: 38 6e GHVVGLFRAAV C TRGVAKAICN.GX004.DQ314805 SEQ ID NO: 39 6f GHAVGIFRAAV C TRGVAKAITH.C-0044.DQ835760 SEQ ID NO: 40 6f GHAVGIFRAAV C TRGVAKAITH.C-0046.DQ835764 SEQ ID NO: 41 6g GHVVGLFRAAV C TRGVAKALHK.HK6554.DQ314806 SEQ ID NO: 42 6g GHVVGLFRAAV C TRGVAKALID.JK046.D63822 SEQ ID NO: 43 6h GHVAGIFRAAV C TRGVAKSL VN.VN004.D84265SEQ ID NO: 44 6i GHVAGIFRAAV C TRGVAKSL TH.C-0159.DQ835762 SEQ ID NO: 456j GHVAGIFRAAV C TRGVAKSL TH.C-0667.DQ835761 SEQ ID NO: 46 6jGHVAGIFRAAV C TRGVAKSL TH.Th553.DQ835769 SEQ ID NO: 47 6k GHVAGIFRAAV CTRGVAKSL CN.KM41.DQ278893 SEQ ID NO: 48 6k GHVAGIFRAAV C TRGVAKSLCN.KM45.DQ278891 SEQ ID NO: 49 6k GHVAGIFRAAV C TRGVAKSL VN.VN405.D84264SEQ ID NO: 50 6l GHVAGIFRAAV C TRGVAKSL US.537796.EF424628 SEQ ID NO: 516m GHAVGVFRAAV C TRGVAKSL TH.C-0185.DQ835765 SEQ ID NO: 52 6mGHAVGVFRAAV C TRGVAKSL TH.C-0208.DQ835763 SEQ ID NO: 53 6n GHVVGIFRAAV CTRGVAKSL CN.KM42.DQ278894 SEQ ID NO: 54 6n GHVVGIFRAAV C TRGVAKSLTH.D86/93.DQ835768 SEQ ID NO: 55 6o GHAVGLFRAAV C TRGVAKAICA.QC227.EF424627 SEQ ID NO: 56 6p GHVVGLFRAAV C TRGVAKAICA.QC216.EF424626 SEQ ID NO: 57 6q GHAVGLFRAAV C TRGVAKAICA.QC99.EF424625 SEQ ID NO: 58 6t GHVVGLFRAAV C TRGVAKAIVN.TV241.EF632069 SEQ ID NO: 59 6t GHVVGLFRAAV C TRGVAKAIVN.TV249.EF632070 SEQ ID NO: 60 6t GHVVGLFRAAV C TRGVAKAIVN.VT21.EF632071 SEQ ID NO: 61 7a SHCVGIFRAAV C TRGVAKAVCA.QC69.EF108306 SEQ ID NO: 62 ^(a)It will be appreciated by one ofordinary skill in the art that every virus is prone to mutation andsubject to polymorphisms, and any genotype consensus sequences describedherein are representative of a given genotype or subtype. Suchrepresentative consensus sequences are available athttp://hcv.lanl.gov/content/sequence/NEWALIGN/align.html.

Drug resistance is emerging as a significant challenge for targetedtherapies. For example, drug resistance has been reported for HCVprotease inhibitors in development. Such compounds include BILN 2061 andVX-950, developed by Boehringer Ingelheim and Vertex Pharmaceuticals,respectively. The structures of BILN 2061 and VX-950 are depicted below.

In fact, a recent article published by Vertex Pharmaceuticals, entitled,“In Vitro Resistance Studies of Hepatitis C Virus Serine Protease,”squarely addresses the problem of mutant resistance observed with VX-950and BILN 2061. See Lin et al., The Journal of Biological Chemistry, Vol.279, No. 17, Issue of April 23, pp. 17508-17514, 2004. This articleconcludes that “future hepatitis C therapy involving small moleculeinhibitors of HCV enzymes might require multidrug combination, as in thecase of the current HIV treatments.” See page 17513, last paragraph.

Resistance to specific antiviral drugs is a major factor limiting theefficacy of therapies against many retroviruses or RNA viruses. Theerror-prone nature of these viruses allows for the development ofmutations that afford resistance to currently available drugs or drugsundergoing clinical testing. The resistance problem is a critical hurdlefaced in drug development of new HCV-specific inhibitors to treat HCVpatients.

A recent in vitro resistance study using two HCV NS3.4A proteaseinhibitors, VX-950 and BILN 2061, found that resistance mutationsselected against either inhibitor resulted in a significant reduction insusceptibility to the inhibitor itself. However, the primary resistancemutations against BILN 2061 were fully susceptible to VX-950, and themajor resistance mutation against VX-950 remained sensitive to BILN 2061(Lin et al., Jour. Biol. Chem. 279(17): 17508-14, 2004).

It has been surprisingly found that provided compounds inhibit at leastfive HCV protease mutants, including A156T, A156S, D168V, and D168A andR155K. This stands in contrast to other known HCV protease inhibitors(e.g., VX-950 and BILN 2061) which inhibit only two mutants each. Infact, no drug described in the prior art has been shown to be aneffective inhibitor of all known HCV protease mutants. For example, andas set forth in Tables 4a and 4b below, where the BILN 2061 and VX-950data are as reported by Lin et al. and elsewhere in the HCV literature,and the data for compound I-3 was obtained according to methods setforth in the Examples, infra. Without wishing to be bound by anyparticular theory, it is believed that compounds of the presentinvention may be effective inhibitors of drug resistant forms of HCVprotease. While Table 4b shows compound I-3 activity against fourreference HCV variants (A156T, A156S, D168V, and D168A), the ensuingexamples will describe other provided compounds of the invention thatare active against these variants as well as a fifth (R155K) variant.

TABLE 4a Comparative K_(i) Values (nM)^(a) BILN 2061 VX-950 WT 19 100A156T >1200 9900 A156S 112 2900 D168V >1200 43 D168A >1200 150^(a)Wild-type data were obtained from cell-based assays, and mutant datawere obtained from biochemical assays. See Lin et al. and protocolsdescribed herein.

TABLE 4b Comparative IC₅₀ Values (nM)^(a) BILN 2061 VX-950 Compound I-1WT 4 402 0.66 A156T — — 3 A156S 7 4650 2 D168V 5090 163 2 D168A 1860 1938 ^(a)Wild-type data were obtained from cell-based assays, and mutantdata were obtained from biochemical assays. See Lin et al. and protocolsdescribed herein.

Without wishing to be bound by any particular theory, it is believedthat a compound of formula I is more effective at inhibiting HCVprotease, or a mutant thereof, as compared to a corresponding compoundof formula I wherein the R³ moiety of formula I is instead a non-warheadgroup, such as straight alkyl (e.g., unsubstituted alkyl), branchedalkyl, cycloalkyl, or alkenyl. For example, a compound of formula I canbe more effective at inhibition of HCV protease, or a mutant thereof, ascompared to a corresponding compound of formula I wherein the R³ moietyof formula I is instead a non-warhead moiety such as methyl, ethyl,propyl, butyl (e.g., t-butyl), unsubstituted straight or branchedalkenyl (e.g. C₁₋₈ alkenyl), cyclohexyl, or cyclopentyl.

A compound of formula I, as disclosed above, can be more potent withrespect to an IC₅₀ against HCV protease, or a mutant such as A156T,A156S, D168V, D168A, or other mutants such as those disclosed herein,than a corresponding compound of formula I wherein the R³ moiety offormula I is instead a non-warhead moiety such as methyl, ethyl, propyl,butyl (e.g., t-butyl), unsubstituted straight or branched alkenyl (e.g.C₁₋₈ alkenyl), cyclohexyl, or cyclopentyl. Such comparative potency of acompound of formula I as compared to a corresponding compound of formulaI wherein the R³ moiety of formula I is instead a non-warhead moiety,can be determined by standard time-dependent assay methods, such asthose described in detail in the Examples section, infra. In certainembodiments, a compound of formula I is measurably more potent than acorresponding compound of formula I wherein the R³ moiety of formula Iis instead a non-warhead moiety such as methyl, ethyl, propyl, butyl(e.g., t-butyl), unsubstituted straight or branched alkenyl (e.g. C₁₋₈alkenyl), cyclohexyl, or cyclopentyl. In some embodiments, a compound offormula I is measurably more potent, wherein such potency is observedafter about 1 minute, about 2 minutes, about 5 minutes, about 10minutes, about 20 minutes, about 30 minutes, about 1 hour, about 2hours, about 3 hours, about 4 hours, about 8 hours, about 12 hours,about 16 hours, about 24 hours, or about 48 hours, than a correspondingcompound of formula I wherein the R³ moiety of formula I is instead anon-warhead moiety such as methyl, ethyl, propyl, butyl (e.g., t-butyl),unsubstituted straight or branched alkenyl (e.g. C₁₋₈ alkenyl),cyclohexyl, or cyclopentyl. In some embodiments, a compound of formula Iis any of about 1.5 times, about 2 times, about 5 times, about 10 times,about 20 times, about 25 times, about 50 times, about 100 times, or evenabout 1000 times more potent than a corresponding compound of formula Iwherein the R³ moiety of formula I is instead a non-warhead moiety suchas methyl, ethyl, propyl, butyl (e.g., t-butyl), unsubstituted straightor branched alkenyl (e.g. C₁₋₈ alkenyl), cyclohexyl, or cyclopentyl.

As used herein, the term “clinical drug resistance” refers to the lossof susceptibility of a drug target to drug treatment as a consequence ofmutations in the drug target

As used herein, the term “resistance” refers to changes in the wild-typenucleic acid sequence coding a target protein, and/or the proteinsequence of the target, which changes decrease or abolish the inhibitoryeffect of the inhibitor on the target protein.

Examples of proteases that are inhibited by the compounds andcompositions described herein and against which the methods describedherein are useful include NS3, NS3•4A, or a mutant thereof.

The activity of a compound utilized in this invention as an inhibitor ofNS3, NS3•4A, or a mutant thereof, may be assayed in vitro, in vivo or ina cell line. In vitro assays include assays that determine inhibition ofeither the serine protease activity and/or the subsequent functionalconsequences, or ATPase activity of activated NS3, NS3•4A, or a mutantthereof. Alternate in vitro assays quantitate the ability of theinhibitor to bind to NS3 or NS3•4A. Inhibitor binding may be measured byradiolabelling the inhibitor prior to binding, isolating theinhibitor/NS3 or inhibitor/NS3•4A complex and determining the amount ofradiolabel bound. Alternatively, inhibitor binding may be determined byrunning a competition experiment where new inhibitors are incubated withNS3 or NS3•4A bound to known radioligands. Detailed conditions forassaying a compound utilized in this invention as an inhibitor of NS3 orNS3•4A, or a mutant thereof, are set forth in the Examples below.

Serine proteases are a large family of proteolytic enzymes that cleavepeptide bonds in proteins. The serine protease family includes thedigestive enzymes chymotrypsin, trypsin, and elastase, and proteasesinvolved in blood clotting. Serine proteases possess a characteristic“catalytic triad” comprising serine, aspartic acid, and histidine, thattogether function to activate serine to form a covalent bond with theenzyme substrate, thereby hydrolyzing a peptide bond. In addition tothose stated above, serine proteases participate in a variety offunctions including immunity and inflammation.

As used herein, the terms “treatment,” “treat,” and “treating” refer toreversing, alleviating, delaying the onset of, or inhibiting theprogress of a disease or disorder, or one or more symptoms thereof, asdescribed herein. In some embodiments, treatment may be administeredafter one or more symptoms have developed. In other embodiments,treatment may be administered in the absence of symptoms. For example,treatment may be administered to a susceptible individual prior to theonset of symptoms (e.g., in light of a history of symptoms and/or inlight of genetic or other susceptibility factors). Treatment may also becontinued after symptoms have resolved, for example to prevent or delaytheir recurrence.

The compounds and compositions, according to the method of the presentinvention, may be administered using any amount and any route ofadministration effective for treating or lessening the severity ofcancer, an autoimmune disorder, a neurodegenerative or neurologicaldisorder, schizophrenia, a bone-related disorder, liver disease, or acardiac disorder. The exact amount required will vary from subject tosubject, depending on the species, age, and general condition of thesubject, the severity of the infection, the particular agent, its modeof administration, and the like. The compounds of the invention arepreferably formulated in dosage unit form for ease of administration anduniformity of dosage. The expression “dosage unit form” as used hereinrefers to a physically discrete unit of agent appropriate for thepatient to be treated. It will be understood, however, that the totaldaily usage of the compounds and compositions of the present inventionwill be decided by the attending physician within the scope of soundmedical judgment. The specific effective dose level for any particularpatient or organism will depend upon a variety of factors including thedisorder being treated and the severity of the disorder; the activity ofthe specific compound employed; the specific composition employed; theage, body weight, general health, sex and diet of the patient; the timeof administration, route of administration, and rate of excretion of thespecific compound employed; the duration of the treatment; drugs used incombination or coincidental with the specific compound employed, andlike factors well known in the medical arts. The term “patient”, as usedherein, means an animal, preferably a mammal, and most preferably ahuman.

The pharmaceutically acceptable compositions of this invention can beadministered to humans and other animals orally, rectally, parenterally,intracisternally, intravaginally, intraperitoneally, topically (as bypowders, ointments, or drops), bucally, as an oral or nasal spray, orthe like, depending on the severity of the infection being treated. Incertain embodiments, the compounds of the invention may be administeredorally or parenterally at dosage levels of about 0.01 mg/kg to about 50mg/kg and preferably from about 1 mg/kg to about 25 mg/kg, of subjectbody weight per day, one or more times a day, to obtain the desiredtherapeutic effect.

In some embodiments, a provided composition is administered to a patientin need thereof once daily. Without wishing to be bound by anyparticular theory, it is believed that prolonged duration of action ofan irreversible inhibitor of HCV NS3 protease is particularlyadvantageous for once daily administration to a patient in need thereoffor the treatment of a disorder associated with HCV NS3 protease. Incertain embodiments, a provided composition is administered to a patientin need thereof at least once daily. In other embodiments, a providedcomposition is administered to a patient in need thereof twice daily,three times daily, or four times daily.

Compounds of formula I, for example, generally provide prolongedduration of action when administered to a patient as compared to acorresponding compound of formula I wherein the R³ moiety of formula Iis instead a non-warhead moiety such as straight alkyl (e.g.,unsubstituted alkyl), branched alkyl, cycloalkyl, or alkenyl. Forexample, a compound of formula I can provide prolonged duration ofaction when administered to a patient as compared to a correspondingcompound of formula I wherein the R³ moiety of formula I is instead anon-warhead moiety such as methyl, ethyl, propyl, butyl (e.g., t-butyl),unsubstituted straight or branched alkenyl (e.g. C₁₋₈ alkenyl),cyclohexyl, or cyclopentyl.

Liquid dosage forms for oral administration include, but are not limitedto, pharmaceutically acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. In addition to the active compounds,the liquid dosage forms may contain inert diluents commonly used in theart such as, for example, water or other solvents, solubilizing agentsand emulsifiers such as ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butylene glycol, dimethylformamide, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor, and sesame oils),glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fattyacid esters of sorbitan, and mixtures thereof. Besides inert diluents,the oral compositions can also include adjuvants such as wetting agents,emulsifying and suspending agents, sweetening, flavoring, and perfumingagents.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

The injectable formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

In order to prolong the effect of a compound of the present invention,it is often desirable to slow the absorption of the compound fromsubcutaneous or intramuscular injection. This may be accomplished by theuse of a liquid suspension of crystalline or amorphous material withpoor water solubility. The rate of absorption of the compound thendepends upon its rate of dissolution that, in turn, may depend uponcrystal size and crystalline form. Alternatively, delayed absorption ofa parenterally administered compound form is accomplished by dissolvingor suspending the compound in an oil vehicle. Injectable depot forms aremade by forming microencapsule matrices of the compound in biodegradablepolymers such as polylactide-polyglycolide. Depending upon the ratio ofcompound to polymer and the nature of the particular polymer employed,the rate of compound release can be controlled. Examples of otherbiodegradable polymers include poly(orthoesters) and poly(anhydrides).Depot injectable formulations are also prepared by entrapping thecompound in liposomes or microemulsions that are compatible with bodytissues.

Compositions for rectal or vaginal administration are preferablysuppositories which can be prepared by mixing the compounds of thisinvention with suitable non-irritating excipients or carriers such ascocoa butter, polyethylene glycol or a suppository wax which are solidat ambient temperature but liquid at body temperature and therefore meltin the rectum or vaginal cavity and release the active compound.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activecompound is mixed with at least one inert, pharmaceutically acceptableexcipient or carrier such as sodium citrate or dicalcium phosphateand/or a) fillers or extenders such as starches, lactose, sucrose,glucose, mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, c) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets and pills, thedosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like. The solid dosage forms of tablets, dragees, capsules, pills,and granules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions that can be usedinclude polymeric substances and waxes. Solid compositions of a similartype may also be employed as fillers in soft and hard-filled gelatincapsules using such excipients as lactose or milk sugar as well as highmolecular weight polethylene glycols and the like.

The active compounds can also be in micro-encapsulated form with one ormore excipients as noted above. The solid dosage forms of tablets,dragees, capsules, pills, and granules can be prepared with coatings andshells such as enteric coatings, release controlling coatings and othercoatings well known in the pharmaceutical formulating art. In such soliddosage forms the active compound may be admixed with at least one inertdiluent such as sucrose, lactose or starch. Such dosage forms may alsocomprise, as is normal practice, additional substances other than inertdiluents, e.g., tableting lubricants and other tableting aids such amagnesium stearate and microcrystalline cellulose. In the case ofcapsules, tablets and pills, the dosage forms may also comprisebuffering agents. They may optionally contain opacifying agents and canalso be of a composition that they release the active ingredient(s)only, or preferentially, in a certain part of the intestinal tract,optionally, in a delayed manner. Examples of embedding compositions thatcan be used include polymeric substances and waxes.

Dosage forms for topical or transdermal administration of a compound ofthis invention include ointments, pastes, creams, lotions, gels,powders, solutions, sprays, inhalants or patches. The active componentis admixed under sterile conditions with a pharmaceutically acceptablecarrier and any needed preservatives or buffers as may be required.Ophthalmic formulation, car drops, and eye drops are also contemplatedas being within the scope of this invention. Additionally, the presentinvention contemplates the use of transdermal patches, which have theadded advantage of providing controlled delivery of a compound to thebody. Such dosage forms can be made by dissolving or dispensing thecompound in the proper medium. Absorption enhancers can also be used toincrease the flux of the compound across the skin. The rate can becontrolled by either providing a rate controlling membrane or bydispersing the compound in a polymer matrix or gel.

According to one embodiment, the invention relates to a method ofinhibiting serine protease activity in a biological sample comprisingthe step of contacting said biological sample with a compound of thisinvention, or a composition comprising said compound.

According to another embodiment, the invention relates to a method ofinhibiting HCV protease, or a mutant thereof, activity in a biologicalsample comprising the step of contacting said biological sample with acompound of this invention, or a composition comprising said compound.In certain embodiments, the invention relates to a method ofirreversibly inhibiting HCV protease, or a mutant thereof, activity in abiological sample comprising the step of contacting said biologicalsample with a compound of this invention, or a composition comprisingsaid compound.

The term “biological sample”, as used herein, includes, withoutlimitation, cell cultures or extracts thereof biopsied material obtainedfrom a mammal or extracts thereof and blood, saliva, urine, feces,semen, tears, or other body fluids or extracts thereof.

Inhibition of HCV protease, or a mutant thereof, activity in abiological sample is useful for a variety of purposes that are known toone of skill in the art. Examples of such purposes include, but are notlimited to, blood transfusion, organ-transplantation, biologicalspecimen storage, and biological assays.

Another embodiment of the present invention relates to a method ofinhibiting HCV protease, or a mutant thereof, activity in a patientcomprising the step of administering to said patient a compound of thepresent invention, or a composition comprising said compound.

According to another embodiment, the invention relates to a method ofinhibiting HCV protease, or a mutant thereof, activity in a patientcomprising the step of administering to said patient a compound of thepresent invention, or a composition comprising said compound. Accordingto certain embodiments, the invention relates to a method ofirreversibly inhibiting HCV protease, or a mutant thereof, activity in apatient comprising the step of administering to said patient a compoundof the present invention, or a composition comprising said compound. Inother embodiments, the present invention provides a method for treatinga disorder mediated by HCV protease, or a mutant thereof, in a patientin need thereof, comprising the step of administering to said patient acompound according to the present invention or pharmaceuticallyacceptable composition thereof. Such disorders are described in detailherein.

Depending upon the particular condition, or disease, to be treated,additional therapeutic agents, which are normally administered to treatthat condition, may be administered in combination with compounds andcompositions of this invention. As used herein, additional therapeuticagents that are normally administered to treat a particular disease, orcondition, are known as “appropriate for the disease, or condition,being treated”.

In certain embodiments, a provided compound, or composition thereof, isadministered in combination with another inhibitor of HCV protease, or avariant thereof. In some embodiments, a provided compound, orcomposition thereof, is administered in combination with anotherantiviral agent. Such antiviral agents include, but are not limited to,immunomodulatory agents, such as α-, β-, and γ-interferons, pegylatedderivatized interferon-α compounds, and thymosin; other anti-viralagents, such as ribavirin, amantadine, and telbivudine; other inhibitorsof hepatitis C proteases (NS2-NS3 inhibitors and NS3-NS4A inhibitors,e.g. BILN 2061 and VX-950); inhibitors of other targets in the HCV lifecycle, including helicase and polymerase inhibitors; inhibitors ofinternal ribosome entry; broad-spectrum viral inhibitors, such as IMPDHinhibitors (e.g., mycophenolic acid and derivatives thereof); orcombinations of any of the above.

In certain embodiments, a combination of 2 or more antiviral agents maybe administered. In certain embodiments, a combination of 3 or moreantiviral agents may be administered. In some embodiments, the antiviralagents are selected from ribavirin or interferon. In other embodiments,the antiviral agent is α-interferon.

Other examples of agents the inhibitors of this invention may also becombined with include, without limitation: treatments for Alzheimer'sDisease such as Aricept® and Excelon®; treatments for HIV such asritonavir; treatments for Parkinson's Disease such as L-DOPA/carbidopa,entacapone, ropinrole, pramipexole, bromocriptine, pergolide,trihexephendyl, and amantadinc; agents for treating Multiple Sclerosis(MS) such as beta interferon (e.g., Avonex® and Rebif®), Copaxone®, andmitoxantrone; treatments for asthma such as albuterol and Singulair®;agents for treating schizophrenia such as zyprexa, risperdal, seroquel,and haloperidol; anti-inflammatory agents such as corticosteroids, TNFblockers, IL-1 RA, azathioprine, cyclophosphamide, and sulfasalazinc;immunomodulatory and immunosuppressive agents such as cyclosporin,tacrolimus, rapamycin, mycophenolate mofetil, interferons,corticosteroids, cyclophophamide, azathioprine, and sulfasalazinc;neurotrophic factors such as acetylcholinesterase inhibitors, MAOinhibitors, interferons, anti-convulsants, ion channel blockers,riluzole, and anti-Parkinsonian agents; agents for treatingcardiovascular disease such as beta-blockers, ACE inhibitors, diuretics,nitrates, calcium channel blockers, and statins; agents for treatingliver disease such as corticosteroids, cholestyramine, interferons, andanti-viral agents; agents for treating blood disorders such ascorticosteroids, anti-leukemic agents, and growth factors; agents thatprolong or improve pharmacokinetics such as cytochrome P450 inhibitors(i.e., inhibitors of metabolic breakdown) and CYP3A4 inhibitors (e.g.,ketokenozole and ritonavir), and agents for treating immunodeficiencydisorders such as gamma globulin.

In certain embodiments, compounds of the present invention, or apharmaceutically acceptable composition thereof, are administered incombination with a monoclonal antibody or an siRNA therapeutic.

Those additional agents may be administered separately from an inventivecompound-containing composition, as part of a multiple dosage regimen.Alternatively, those agents may be part of a single dosage form, mixedtogether with a compound of this invention in a single composition. Ifadministered as part of a multiple dosage regime, the two active agentsmay be submitted simultaneously, sequentially or within a period of timefrom one another normally within five hours from one another.

As used herein, the term “combination,” “combined,” and related termsrefers to the simultaneous or sequential administration of therapeuticagents in accordance with this invention. For example, a compound of thepresent invention may be administered with another therapeutic agentsimultaneously or sequentially in separate unit dosage forms or togetherin a single unit dosage form. Accordingly, the present inventionprovides a single unit dosage form comprising a compound of formula I,an additional therapeutic agent, and a pharmaceutically acceptablecarrier, adjuvant, or vehicle.

The amount of both, an inventive compound and additional therapeuticagent (in those compositions which comprise an additional therapeuticagent as described above) that may be combined with the carriermaterials to produce a single dosage form will vary depending upon thehost treated and the particular mode of administration. Preferably,compositions of this invention should be formulated so that a dosage ofbetween 0.01-100 mg/kg body weight/day of an inventive can beadministered.

In those compositions which comprise an additional therapeutic agent,that additional therapeutic agent and the compound of this invention mayact synergistically. Therefore, the amount of additional therapeuticagent in such compositions will be less than that required in amonotherapy utilizing only that therapeutic agent. In such compositionsa dosage of between 0.01-100 mg/kg body weight/day of the additionaltherapeutic agent can be administered.

The amount of additional therapeutic agent present in the compositionsof this invention will be no more than the amount that would normally beadministered in a composition comprising that therapeutic agent as theonly active agent. Preferably the amount of additional therapeutic agentin the presently disclosed compositions will range from about 50% to100% of the amount normally present in a composition comprising thatagent as the only therapeutically active agent.

EXEMPLIFICATION

As depicted in the Examples below, in certain exemplary embodiments,compounds are prepared according to the following general procedures. Itwill be appreciated that, although the general methods depict thesynthesis of certain compounds of the present invention, the followinggeneral methods, and other methods known to one of ordinary skill in theart, can be applied to all compounds and subclasses and species of eachof these compounds, as described herein.

Compound numbers utilized in the Examples, below, correspond to compoundnumbers set forth in Table 3, supra.

Example 1

The title compound was prepared according to the steps and intermediatesas described below.

Step 1a: Intermediate 1a

To a solution of (1R,2S)-1-amino-2-vinylcyclopropane carboxylic acidethyl ester toluenesulfonic acid (0.33 g, 1.0 mmol) and(2S,4R)-1-(tert-butoxycarbonyl)-4-(4-fluoroisoindoline-2-carbonyloxy)pyrrolidine-2-carboxylicacid (0.4 g, 1.0 mmol) in 10 mL of acetonitrile was added HATU (0.44 g,1.2 mmol) and then DIEA (0.46 mL, 2.5 mmol) under stirring. The mixturewas stirred at r.t. for two hours. After the complete consumption ofstarting materials, the reaction mixture was evaporated. The residue wasdissolved in 30 mL ethyl acetate and washed with water and brine twiceand dried over Na₂SO₄. After removal of solvent, the crude product wassubject to chromatography on silica gel (hexane:EtOAc=1:1). 0.35 g ofthe title compound was obtained: MS m/z: 532.0 (M+H⁺).

Step 1b: Intermediate 1b

To a solution of the product of step 1a (0.35 g, 0.66 mmol) in 5 mL ofTHF/MeOH (1:1) was added 1N LiOH aqueous solution (2 mL, 2.0 mmol).After stirring at r.t. for 10 hours, the reaction mixture wasneutralized with 1.0 N HCl. The organic solvents were evaporated undervacuum, and the remaining aqueous phase was acidified to pH-3 using 1.0N HCl and was extracted with EtOAc. The organic layer was washed withbrine, and was dried over anhydrous magnesium sulfate. After removal ofsolvent, 0.3 g of the title compound was obtained: MS m/z: 526.2(M+Na⁺).

Step 1c: Intermediate 1c

To a solution of the product of step 1b (0.30 g, 0.6 mmol) in 10 mL ofDCM was added CDI (0.16 g, 1.0 mmol) and the resulting solution wasstirred at 40° C. for 1 hour. cyclopropylsulfonamide (0.18 g, 1.5 mmol)and DBU (0.16 g, 1.0 mmol) were added to the reaction mixture. Themixture was stirred at 40° C. for additional 10 hours. The solvent wasthen removed and the residue was diluted with EtOAc and was washed withaqueous NaOAc buffer (pH˜5, 2×10 mL), NaHCO₃ solution and brine. Afterdrying over Na₂SO₄ and removal of solvent, the residue was subjected tochromatography on silica gel using hexane/EtOAc (1:1˜1:2). A total of0.30 g of the title compound was obtained: R_(f) 0.1 (EtOAc:hexane=1:1),MS m/z: 605.0 (M−1).

The product from step 1c (0.25 g, 0.41 mmol) was dissolved in 4 N HCl indioxane. The mixture was stirred at r.t. for 1 hour. After removal ofsolvents, a 10-mL portion of DCM was poured in followed by evaporationto dryness. This process of DCM addition followed by evaporation wasrepeated four times to give a residue solid which was used directly forthe next step: MS m/z: 507.0 (M+H⁺).

Step 1e: Intermediate 1e

To a solution of the product of step 1d (0.16 g, 0.28 mmol) andN-Boc-3-(Fmoc)amino-L-alanine (0.15 g, 0.35 mmol) in 5.0 mL of DMF wasadded HATU (125 mg, 0.33 mmol) and DIEA (130 mg, 1.0 mmol) at r.t. understirring. TLC analysis indicated completion of the coupling reaction hadoccurred after one hour. A 20-mL portion of EtOAc was poured in and themixture was washed with a buffer (pH˜4, AcONa/AcOH), NaHCO₃ and brine,and was dried over MgSO₄. After removal of solvent, the crude oilproduct was subject to chromatography on silica gel (eluents:EtOAc/hexane). A total of 0.14 g of the title compound was obtained.

Step 1f: Intermediate 1f

A solution of 0.10 g of the product of step 1e in 1 mL of DMF with 12%piperidine was stirred for 1.5 hours at r.t. and then was evaporated todryness under high vacuum. The residue was trituated with hexane/ether(4:1) to yield 70 mg of the title compound.

Step 1g: Compound (I-1)

(3R,5S)-1-(S)-3-acrylamido-2-(tert-butoxycarbonylamino)propanoyl)-5-((1R,2S)-1-(cyclopropylsulfonylcarbamoyl)-2-vinylcyclopropylcarbamoyl)pyrrolidin-3-yl4-fluoroisoindoline-2-carboxylate

Acryloyl chloride (10 uL, 0.12 mmol) was added dropwise at 0° C. to astirred solution of 55 mg (0.08 mmol) of the product from step 1f in 3mL of DCM containing 3 equiv. of triethylamine. The reaction mixture wasstirred at r.t. for 1.5 hrs and then was diluted with 10 mL of DCM. Theresulting solution was washed twice with brine and was dried overmagnesium sulfate. Removal of solvent afforded the crude product, whichwas purified by chromatography on silica gel eluting first withhexane/EtOAc (1:3˜1:5) and then with DCM-methanol (50:1˜25:1)). A totalof 27 mg of the title compound was obtained: R_(f) 0.4 (EA:MeOH=10:1);MS m/z: 746.9 (M+H⁺).

In similar fashion using the product of Intermediate 1f the followingcompounds were prepared:

(3R,5S)-1-(S)-2-(tert-butoxycarbonylamino)-3-(vinylsulfonamido)propanoyl)-5-((1R,2S)-1-(cyclopropylsulfonylcarbamoyl)-2-vinylcyclopropylcarbamoyl)pyrrolidin-3-yl-4-fluoroisoindoline-2-carboxylate

Rf: 0.50 (EtOAc/MeOH 10:1); MS m/z: 805.3 (M+H⁺).

(3R,5S)-1-((2S)-2-(tert-butoxycarbonylamino)-3-(2-chloro-2-phenylacetamido)propanoyl)-5-((1R,2S)-1-(cyclopropylsulfonylcarbamoyl)-2-vinylcyclopropylcarbamoyl)pyrrolidin-3-yl-4-fluoroisoindoline-2-carboxylate

Rf: 0.50 (DCM/MeOH 95:5); MS m/z: 845.2 (M+H⁺).

(3R,5S)-1-((S)-2-(tert-butoxycarbonylamino)-3-((E)-4-(dimethylamino)but-2-enamido)propanoyl)-5-(1R,2S)-1-(cyclopropylsulfonylcarbamoyl)-2-vinylcyclopropylcarbamoyl)pyrrolidin-3-yl-4-fluoroisoindoline-2-carboxylate

Rf: 0.35 (DCM/MeOH 9:1); MS m/z: 804.3 (M+H⁺).

In similar fashion using the product of Intermediate 1d and(S)-4-(Fmocamino)-2-(tert-butoxycarbonylamino)butanoic acid, thefollowing compound was prepared:

(3R,5S)-1-((S)-4-acrylamido-2-(tert-butoxycarbonylamino)butanoyl)-5-((1R,2S)-1-(cyclopropylsulfonylcarbamoyl)-2-vinylcyclopropylcarbamoyl)pyrrolidin-3-yl4-fluoroisoindoline-2-carboxylate

Rf: 0.40 (EtOAc/MeOH 10:1); MS m/z: 761.3 (M+H⁺).

Example 2

The title compound was prepared according to the steps and intermediatesas described below.

Step 2a: Intermediate 2a

To a solution of the product of step 1d from Example 1 (0.12 g, 0.22mmol) and N-Boc-glycine (0.054 g, 0.31 mmol) in 4.0 mL of acetonitrilewas added HATU (133 mg, 0.35 mmol) and DIEA (0.12 mL, 0.66 mmol) at r.t.under stirring. The reaction mixture was stirred for 2 h. LC-MS and TLCanalysis indicated completion of the coupling reaction. A 20-mL of EtOAcwas poured in and the mixture was washed with a buffer (pH˜4,AcONa/AcOH), NaHCO₃ and brine, and was dried over Na₂SO₄. After removalof solvent, the crude product was subject to chromatography on silicagel (eluents: EtOAc/hexane). A total of 0.10 g of the title compound wasobtained: R_(f) 0.2 (EtOAc); MS m/z: 664.0 (M+H⁺).

Step 2b: Intermediate 2b

The product from step 2a (0.10 g, 0.15 mmol) was dissolved in 2 mL of 4N HCl in dixoxane and the reaction was stirred for 1 hour at RT. Afterremoval of solvents, a 3-mL portion of DCM was poured in followed byevaporation to dryness. This process of DCM addition followed byevaporation was repeated three times to give the title compoundIntermediate 2b as its HCl salt (0.10 g). MS m/z: 564.0 (M+H⁺).

Step 2c:

(3R,5S)-1-(2-acrylamidoacetyl)-5-((1R,2S)-1-(cyclopropylsulfonylcarbamoyl)-2-vinylcyclopropylcarbamoyl)pyrrolidin-3-yl4-fluoro isoindoline-2-carboxylate

The title compound was made by coupling Intermediate 2b and acrylic acidusing HATU following the coupling reactions described for Intermediate2a. A total of 50 mg of the title compound was obtained: R_(f) 0.1(EtOAc); MS m/z: 617.9 (M+H⁺).

Following the procedures described in Example 2, the following compoundswere made similarly:

(3R,5S)-1-(2-(E)-but-2-enamidoacetyl)-5-((1R,2S)-1-(cyclopropylsulfonylcarbamoyl)-2-vinylcyclopropylcarbamoyl)pyrrolidin-3-yl-4-fluoroisoindoline-2-carboxylate

MS m/z: 632.0 (M+H⁺).

(3R,5S)-1-((R)-2-acrylamidopropanoyl)-5-((1R,2S)-1-(cyclopropylsulfonylcarbamoyl)-2-vinylcyclopropylcarbamoyl)pyrrolidin-3-yl-4-fluoroisoindoline-2-carboxylate

MS m/z: 632.1 (M+H⁺).

(3R,5S)-1-((R)-2-acrylamido-3-methylbutanoyl)-5-((1R,2S)-1-(cyclopropylsulfonylcarbamoyl)-2-vinylcyclopropylcarbamoyl)pyrrolidin-3-yl-4-fluoroisoindoline-2-carboxylate

MS m/z: 660.2 (M+H⁺).

(3R,5S)-1-(2-(2-acetoxybenzamido)acetyl)-5-((1R,2S)-1-(cyclopropylsulfonylcarbamoyl)-2-vinylcyclopropylcarbamoyl)pyrrolidin-3-yl-4-fluoroisoindoline-2-carboxylate

MS m/z: 724.0 (M+H⁺).

(5S)-1-(2)-2-(2-chloropyrimidin-4-ylamino)propanoyl)-5-((1R,2S)-1-(cyclopropylsulfonylcarbamoyl)-2-vinylcyclopropylcarbamoyl)pyrrolidin-3-yl-4-fluoroisoindoline-2-carboxylate

Rf: 0.35 (DCM/MeOH 95:5), MS m/z: 690.3 (M+H⁺).

Following the procedures described in Example 2, the following compoundswere made similarly:

(5S)-1-(2-acetamido-2-(1-acryloylazetidin-3-yl)acetyl)-5-((1R,2S)-1-(cyclopropylsulfonylcarbamoyl)-2-vinylcyclopropylcarbamoyl)pyrrolidin-3-yl-4-fluoroisoindoline-2-carboxylate

MS m/z: 715.2 (M+H⁺).

(5S)-1-(2-(1-acryloylazetidin-3-yl)-2-(cyclopentyloxycarbonylamino)acetyl)-5-((1R,2S)-1-(cyclopropylsulfonylcarbamoyl)-2-vinylcyclopropylcarbamoyl)pyrrolidin-3-yl-4-fluoroisoindoline-2-carboxylate

MS m/z: 785.2 (M+H⁺).

Example 3

The title compound was prepared according to the steps and intermediatesas described below.

Step 3a: Intermediate 3a

To a solution of (S)-3-amino-2-(tert-butoxycarbonylamino)propanoic acid(2.04 g, 10 mmol), TEA (4.5 mL, 30 mmol) in 50 mL CH₂Cl₂ was addednitrobenzenesulfonyl chloride (2.9 g, 13.0 mmol) at RT. The mixture wasstirred for 10 hours at RT. The solvent was removed under vacuumfollowed by the addition of 100 mL EtOAc. The organic layer was washedwith 1 N HCl (to pH 3), water and brine. The organic layer was driedover Na₂SO₄, filtered and the solvent was removed to afford the crudeIntermediate 3a (4.0 g).

Step 3b: Intermediate 3b

The crude Intermediate 3a (2.0 g), K₂CO₃ (1.5, 4 equiv.) were dissolvedin 10 mL DMF. MeI (0.8 mL, 4 eqiv.) was added to the reaction at RT. Theresulting mixture was stirred for 20 hours. The DMF was mostly removedunder vacuum and 100 mL EtOAc was added and the mixture was washed withwater and brine. The organic layer was dried over Na₂SO₄. After removalof solvent, the crude product was subject to a short silica gel column(eluents: EtOAc/hexane) to produce 1.62 g of the Intermediate 3b. MSm/z: 439.9 (M+Na⁺).

Step 3c: Intermediate 3c

To a solution of Intermediate 3b (1.6 g, 3.8 mmol) in 10 mL of THF/MeOH(1:1) was added 1 N LiOH aqueous solution (5.8 mL, 5.8 mmol). Afterstirring at r.t. for 10 hours, the reaction mixture was neutralized with1.0 N HCl. The organic solvent was evaporated under vacuum, and theremaining aqueous phase was acidified to pH˜3 using 1.0 N HCl and wasextracted with EtOAc. The organic layer was washed with brine, and wasdried over anhydrous sodium sulfate. After removal of solvent, 1.5 g ofIntermediate 3c was obtained. MS m/z: 402.0 (M−1, negative mode).

Step 3d: Intermediate 3d

To a solution of Intermediate 1d (0.12 g, 0.20 mmol) and Intermediate 3c(0.12 g, 0.3 mmol) in 5.0 mL of anhydrous acetonitrile was added HATU(0.11 g, 0.3 mmol) and DIEA (0.14 mL, 0.9 mmol) at r.t. under stirring.TLC analysis and LC-MS indicated completion of the coupling reactionafter one hour. A 20-mL portion of EtOAc was poured in and the mixturewas washed with a buffer (pH˜4, AcONa/AcOH), NaHCO₃ and brine. Theorganic layer was dried over Na₂SO₄. After removal of solvent, the crudeproduct was subject to chromatography on silica gel (eluents:EtOAc/hexane). A total of 0.10 g of Intermediate 3d was obtained: R_(f)0.1 (EtOAc); MS m/z: 891.8 (M+H⁺).

Step 3e: Intermediate 3e

To a solution of Intermediate 3d (0.10 g, 0.11 mmol) in 3 mL DMF wasadded phenylthiol (30 mg, 0.26 mmol) and K₂CO₃ (40 mg, 0.3 mmol). Theresulting mixture was stirred for 20 hours at RT. 30 mL EtOAc was addedand the mixture was washed with water and brine and water. The organiclayer was dried over Na₂SO₄. After removal of solvent, the crude productwas subject to chromatography on silica gel (eluents: EtOAc/hexane) toproduce 0.1 g of crude Intermediate 3e. MS m/z: 706.9 (M+H⁺).

Step 3f: Compound I-11

(3R,5S)-1-(S)-2-(tert-butoxycarbonylamino)-3-(N-methylacrylamido)propanoyl)-5-((1R,2S)-1-(cyclopropylsulfonylcarbamoyl)-2-vinylcyclopropylcarbamoyl)pyrrolidin-3-yl4-fluoroisoindoline-2-carboxylate (I-11)

Acryloyl chloride (9 uL, 0.11 mmol) was added dropwise at 0° C. to astirred solution of 0.1 g (0.1 mmol) of the product from step 3e in 3 mLof DCM containing 0.04 mL (0.3 mmol) of triethylamine. The reactionmixture was stirred at r.t. for 1.5 hrs and then was diluted with 10 mLof DCM. The resulting solution was washed twice with brine and was driedover magnesium sulfate. Removal of solvent afforded the crude product,which was purified by chromatography on silica gel eluting first withhexane/EtOAc (1:3˜1:5) and then with EtOAc. A total of 20 mg of thetitle compound was obtained:

R_(f) 0.15 (EtOAc); MS m/z: 760.9 (M+H⁺). ¹HNMR (CD₃OD, 400 MHz) δ 7.32(m, 1H), 7.13-6.98 (m, 2H), 6.75 (m, 1H), 6.23 (dd, 1H, J=2.3, 16.5 Hz),5.73 (m, 2H), 5.45-5.29 (m, 2H), 5.12 (dd, 1H, J=1.4, 10.0 Hz), 4.72 (s,4H), 4.45 (m, 1H), 4.25-4.09 (m, 1H), 3.91 (m, 1H), 3.75-3.50 (m, 1H),3.15 (s, 3H), 2.96 (m, 1H), 2.42 (m, 1H), 2.25 (m, 2H), 1.87 (m, 1H),1.45-0.85 (m, 14H).

In similar fashion using the Intermediate 3e, 2-chloroethanesulfonylchloride, and triethyl amine, the following compound was prepared:

(3R,5S)-1-((S)-2-(tert-butoxycarbonylamino)-3-(N-methylvinylsulfonamido)propanoyl)-5-(1R,2S)-1-(cyclopropylsulfonylcarbamoyl)-2-vinylcyclopropylcarbamoyl)pyrrolidin-3-yl-4-fluoroisoindoline-2-carboxylate

Rf: 0.55 (DCM/MeOH 95:5); MS m/z: 797.3 (M+H⁺).

In similar fashion, using(S)-4-(Fmocamino)-2-(tert-butoxycarbonylamino)butanoic acid in step 3ain the place of (S)-3-amino-2-(tert-butoxycarbonylamino)propanoic acid,the following compound was prepared:

(3R,5S)-1-(S)-2-(tert-butoxycarbonylamino)-4-(N-methylacrylamido)butanoyl)-5-((1R,2S)-1-(cyclopropylsulfonylcarbamoyl)-2-vinylcyclopropylcarbamoyl)pyrrolidin-3-yl4-fluoroisoindoline-2-carboxylate

Rf: 0.45 (DCM/MeOH 95:5); MS m/z: 775.3 (M+H⁺).

In similar fashion, following the procedures described in Example 3,compound I-14 can be made by using ethyl iodide in step 3b in place ofmethyl iodide:

(3R,5S)-1-(S)-2-(tert-butoxycarbonylamino)-3-(N-ethylacrylamido)propanoyl)-5-(1R,2S)-1-(cyclopropylsulfonylcarbamoyl)-2-vinylcyclopropylcarbamoyl)pyrrolidin-3-yl-4-fluoroisoindoline-2-carboxylate

Compound I-15 was made by following the procedures described in Example3, using allyl bromide in step 3b in place of methyl iodide.

(3R,5S)-1-(S)-3-(N-allylacrylamido)-2-(tert-butoxycarbonylamino)propanoyl)-5-((1R,2S)-1-(cyclopropylsulfonylcarbamoyl)-2-vinylcyclopropylcarbamoyl)pyrrolidin-3-yl-4-fluoroisoindoline-2-carboxylate

Rf: 0.58 (DCM/MeOH 95:5); MS m/z: 787.3 (M+H⁺).

The following compounds can be made by starting with the(1R,2S)-1-amino-2-ethylcyclopropane carboxylic acid ethyl ester in step1a and following the appropriate procedures described in Example 3:

(3R,5S)-1-(S)-3-(N-allylacrylamido)-2-(tert-butoxycarbonylamino)propanoyl)-5-((1R,2R)-1-(cyclopropylsulfonylcarbamoyl)-2-ethylcyclopropylcarbamoyl)pyrrolidin-3-yl-4-fluoroisoindoline-2-carboxylate

(3R,5S)-1-((S)-2-(tert-butoxycarbonylamino)-3-(N-methylacrylamido)propanoyl)-5-((1R,2R)-1-(cyclopropylsulfonylcarbamoyl)-2-ethylcyclopropylcarbamoyl)pyrrolidin-3-yl4-fluoroisoindoline-2-carboxylate

(3R,5S)-1-((S)-3-acrylamido-2-(tert-butoxycarbonylamino)propanoyl)-5-((1R,2R)-1-(cyclopropylsulfonylcarbamoyl)-2-ethylcyclopropylcarbamoyl)pyrrolidin-3-yl-4-fluoroisoindoline-2-carboxylate

In similar fashion, the following compound was prepared:

(5S)-1-(2-(tert-butoxycarbonylamino)-3-(E)-4-(dimethylamino)-N-methylbut-2-enamido)propanoyl)-5-(1R,2S)-1-(cyclopropylsulfonylcarbamoyl)-2-vinylcyclopropylcarbamoyl)pyrrolidin-3-yl-4-fluoroisoindoline-2-carboxylate

Rf: 0.45 (DCM/MeOH 95:5); MS m/z: 818.5 (M+H⁺).

Example 4

The title compound was prepared according to the steps and intermediatesas described below.

Step 4a: Intermediate 4a

The Intermediate 1e from step 1e was treated with 4 N HCl according tothe procedure described in step 1d to afford the Intermediate 4a as itsHCl salt.

MS m/z: 815.2 (M+H⁺).

Step 4b: Intermediate 4b

Cyclopentylchloroformate (1.5 equiv.) was added dropwise at 0° C. to astirred solution of Intermediate 4a (1 equiv.) from step 4a in DCMcontaining 3 equiv. of triethylamine. The reaction mixture was stirredat r.t. for 1.5 hrs and then was diluted with 10 mL of DCM. Theresulting solution was washed twice with brine and was dried overmagnesium sulfate. Removal of solvent afforded the crude product, whichwas purified by chromatography on silica gel eluting first withhexane/EtOAc (1:3˜1:5) and then with EtOAc to afford the title compound(60-90%): MS m/z: 925.2 (M−1).

Step 4c: Compound I-19

(3R,5S)-1-(S)-3-acrylamido-2-(cyclopentyloxycarbonylamino)propanoyl)-5-((1R,2S)-1-(cyclopropylsulfonylcarbamoyl)-2-vinylcyclopropylcarbamoyl)pyrrolidin-3-yl4-fluoroisoindoline-2-carboxylate

The title compound was prepared from Intermediate 4b according to theprocedures described in step 1f and step 1g. MS m/z: 759.0 (M+H⁺).

Starting from the appropriate intermediates, in similar fashion, thefollowing compounds were prepared:

(3R,5S)-1-((S)-2-(cyclopentyloxycarbonylamino)-3-(N-methylacrylamido)propanoyl)-5-((1R,2S)-1-(cyclopropylsulfonylcarbamoyl)-2-vinylcyclopropylcarbamoyl)pyrrolidin-3-yl-4-fluoroisoindoline-2-carboxylate

Rf: 0.4 (EtOAc/MeOH 20:1); MS m/z: 773.2 (M+H⁺).

(3R,5S)-1-((S)-2-(cyclopentyloxycarbonylamino)-4-(N-methylacrylamido)butanoyl)-5-(1R,2S)-1-(cyclopropylsulfonylcarbamoyl)-2-vinylcyclopropylcarbamoyl)pyrrolidin-3-yl-4-fluoroisoindoline-2-carboxylate

Rf: 0.4 (EtOAc/MeOH 20:1); MS m/z: 787.3 (M+H⁺).

(5S)-5-((1R,2S)-1-(cyclopropylsulfonylcarbamoyl)-2-vinylcyclopropylcarbamoyl)-1-(2-(4-(methoxycarbonyl)bicyclo[2.2.2]octane-1-carboxamido)-3-(N-methylacrylamido)propanoyl)pyrrolidin-3-yl-4-fluoroisoindoline-2-carboxylate

Rf: 0.45 (EtOAc/MeOH 10:1); MS m/z: 855.3 (M+H⁺).

Example 5

The title compound was prepared according to the steps and intermediatesas described below:

Step 5a: Intermediate 5a

To a solution of Boc-L-Threonine (0.44 g 2.0 mmol) in 10.0 mL of DCM wasadded crotyl chloride (0.32 g, 3.0 mmol) at RT followed by the additionof catalytic amount of DMAP and TEA (1.0 mL, 6 mmol). The reactionmixture was stirred for 10 h at RT. Aqueous NaHCO₃ solution (10 mL) wasadded to quench the reaction. After 2 hours, 1 N HCl aqueous solutionwas added slowly to pH ˜3. The DCM layer was collected and the aqueouswas extracted by DCM (2×10 mL). The organic layer was dried over Na₂SO₄,filtered and the solvent was removed to provide the crude product.

Step 5b: I-22

(3R,5S)-1-((2S,3R)-3-(E)-but-2-enoyloxy)-2-(tert-butoxycarbonylamino)butanoyl)-5-(1R,2S)-1-(cyclopropylsulfonylcarbamoyl)-2-vinylcyclopropylcarbamoyl)pyrrolidin-3-yl-4-fluoroisoindoline-2-carboxylate

The title compound was made by coupling Intermediate 1d from Example 1and Intermediate 5a using HATU following the coupling reactionsdescribed for Intermediate 1e in Example 1. A total of 90 mg of thetitle compound was obtained from 109 mg of Intermediate 1d: R_(f) 0.5(EtOAc); MS m/z: 774.3 (M+H⁺).

Starting from the Intermediate 1d, by coupling with the appropriateintermediates, in similar fashion, the following compounds wereprepared:

(3R,5S)-1-((S)-2-(tert-butoxycarbonylamino)-3-(2-(tert-butoxycarbonylamino)acryloyloxy)propanoyl)-5-((1R,2S)-1-(cyclopropylsulfonylcarbamoyl)-2-vinylcyclopropylcarbamoyl)pyrrolidin-3-yl-4-fluoroisoindoline-2-carboxylate

MS m/z: 862.2 (M−1).

(3R,5S)-1-((2S,3R)-3-(acryloyloxy)-2-(tert-butoxycarbonylamino)butanoyl)-5-((1R,2S)-1-(cyclopropylsulfonylcarbamoyl)-2-vinylcyclopropylcarbamoyl)pyrrolidin-3-yl-4-fluoroisoindoline-2-carboxylate

Rf: 0.4 (EtOAc); MS m/z: 760.1 (M−1).

(3R,5S)-1-(S)-2-(tert-butoxycarbonylamino)-3-(2E,4E)-hexa-2,4-dienoyloxy)propanoyl)-5-(1R,2S)-1-(cyclopropylsulfonylcarbamoyl)-2-vinylcyclopropylcarbamoyl)pyrrolidin-3-yl-4-fluoroisoindoline-2-carboxylate

MS m/z: 786.1 (ES−).

Example 6 Compound I-25

(I-25): The title compound was prepared according to the steps andintermediates as described below:

Step 6a: Intermediate 6a

To a solution of N-Boc-pyroglutamic acid (0.23 g 1.0 mmol) in 10.0 mL ofanhydrous THF was added 2-methylprop-1-enyl)magnesium bromide (0.5 M inTHF, 5 mL, 2.5 mmol) at −78° C. slowly. The reaction mixture was stirredfor 1 h at −78° C. 1 N HCl (2.5 mL) aqueous solution was added and themixture was slowly warmed up to RT. The pH was adjusted to ˜3-4 by 1 NHCl. The THF was then removed under vacuum and the remaining aqueous wasextracted by DCM (3×15 mL). The organic layer was dried over Na₂SO₄,filtered and the solvent was removed to provide the crude product.

Step 6b: I-25

(3R,5S)-1-((S)-2-(tert-butoxycarbonylamino)-7-methyl-5-oxooct-6-enoyl)-5-((1R,2S)-1-(cyclopropylsulfonylcarbamoyl)-2-vinylcyclopropylcarbamoyl)pyrrolidin-3-yl-4-fluoroisoindoline-2-carboxylate

The title compound was made by coupling Intermediate 1d from Example 1and Intermediate 6a using HATU following the coupling reactionsdescribed for Intermediate 1e in Example 1. A total of 80 mg of thetitle compound was obtained from 108 mg of Intermediate 1d: R_(f) 0.3(EtOAc); MS m/z: 774.1 (M+H⁺). ¹HNMR (CD₃OD, 400 MHz) δ 7.31 (dd, 1H,J=13.3, 7.4 Hz), 7.13-6.98 (m, 2H), 6.18 (s, 1H), 5.74 (m, 1H), 5.38 (s,1H), 5.32 (d, 1H, J=17.0 Hz), 5.12 (d, 1H, J=10.1 Hz), 4.72 (s, 4H),4.48 (dd, 1H, J=17.0, 9.16 Hz), 4.29 (m, 2H), 3.89 (m, 1H), 2.93 (m,1H), 2.60-2.35 (m, 2H), 2.22 (m, 2H), 2.10 (s, 3H), 2.02-1.75 (br, 1H),1.88 (s, 3H), 1.46-0.80 (m, 14H).

¹³C NMR (CD₃OD, 100 MHz): δ 201.8, 175.3, 174.5, 170.6, 157.7, 156.9,155.6, 141.1, 134.2, 131.2, 124.8, 119.9, 119.7, 118.6, 115.0, 114.8,80.3, 76.1, 61.0, 55.0, 54.9, 54.8, 53.5, 53.3, 52.7, 50.3, 50.1, 42.6,40.3, 35.7, 35.4, 32.1, 28.7, 28.5, 27.7, 26.9, 24.0, 20.9, 6.74, 6.47.

Starting from the Intermediate 1d, by coupling with the appropriateintermediates made similarly as described in Step 6a, the followingcompounds were prepared:

(3R,5S)-1-((S,E)-2-(tert-butoxycarbonylamino)-5-oxooct-6-enoyl)-5-((1R,2S)-1-(cyclopropylsulfonylcarbamoyl)-2-vinylcyclopropylcarbamoyl)pyrrolidin-3-yl-4-fluoroisoindoline-2-carboxylate

A total of 80 mg of the title compound was obtained from 150 mg ofIntermediate 1d: R_(f) 0.3 (EtOAc); MS m/z: 760.3 (M+H⁺).

(3R,5S)-1-((S)-2-(tert-butoxycarbonylamino)-6-methyl-5-oxohept-6-enoyl)-5-((1R,2S)-1-(cyclopropylsulfonylcarbamoyl)-2-vinylcyclopropylcarbamoyl)pyrrolidin-3-yl4-fluoroisoindoline-2-carboxylate: A total of 80 mg of the titlecompound was obtained from 150 mg of Intermediate 1d

R_(f) 0.4 (EtOAc); MS m/z: 782.2 (M+Na⁺). ¹HNMR (CD₃OD, 400 MHz) δ 7.31(dd, 1H, J=13.3, 7.4 Hz), 7.09 (dd, 1H, J=33, 7.4 Hz), 7.0 (m, 1H), 6.12(s, 1H), 5.82 (s, 1H), 5.74 (m, 1H), 5.39 (s, 1H), 5.31 (dd, 1H, J=1.4,17.0 Hz), 5.12 (dd, 1H, J=10.1, 1.4 Hz), 4.73 (m, 4H), 4.48 (m, 1H),4.32 (m, 2H), 3.90 (m, 1H), 2.91 (m, 1H), 2.42 (m, 1H), 2.22 (m, 2H),2.01 (m, 1H), 1.90-1.85 (m, 2H), 1.84 (s, 3H), 1.40-1.02 (m, 14H).

(3R,5S)-1-((S,E)-2-(tert-butoxycarbonylamino)-6-methyl-5-oxooct-6-enoyl)-5-((1R,2R)-1-(cyclopropylsulfonylcarbamoyl)-2-ethylcyclopropylcarbamoyl)pyrrolidin-3-yl4-fluoroisoindoline-2-carboxylate

A total of 50 mg of the title compound was obtained from 150 mg ofIntermediate 1d: R_(f) 0.5 (EtOAc); MS m/z: 796.2 (M+Na⁺).

(3R,5S)-1-((S)-2-(tert-butoxycarbonylamino)-5-methyl-4-oxohex-5-enoyl)-5-((1R,2S)-1-(cyclopropylsulfonylcarbamoyl)-2-vinylcyclopropylcarbamoyl)pyrrolidin-3-yl4-fluoroisoindoline-2-carboxylate

MS m/z: 768.2 (M+Na^(|)).

(3R,5S)-1-((S)-2-(tert-butoxycarbonylamino)-5-methyl-4-oxohex-5-enoyl)-5-((1R,2R)-1-(cyclopropylsulfonylcarbamoyl)-2-ethylcyclopropylcarbamoyl)pyrrolidin-3-yl4-fluoroisoindoline-2-carboxylate

MS m/z: 770.3 (M+Na⁺).

(3R,5S)-1-((S,E)-2-(tert-butoxycarbonylamino)-5-methyl-4-oxohept-5-enoyl)-5-((1R,2S)-1-(cyclopropylsulfonylcarbamoyl)-2-vinylcyclopropylcarbamoyl)pyrrolidin-3-yl4-fluoroisoindoline-2-carboxylate

MS m/z: 782.3 (M+Na⁺).

(3R,5S)-1-((S,Z)-2-(tert-butoxycarbonylamino)-8,8-dimethyl-5-oxonon-6-enoyl)-5-((1R,2S)-1-(cyclopropylsulfonylcarbamoyl)-2-vinylcyclopropylcarbamoyl)pyrrolidin-3-yl4-fluoroisoindoline-2-carboxylate

¹HNMR (CD₃OD, 400 MHz) δ 7.3 (m, 1H), 7.14-6.99 (m, 2H), 6.03 (d, 1H,J=12.8 Hz), 5.85 (d, 1H, J=13.0 Hz), 5.76 (m, 1H), 5.40 (s, 1H), 5.32(d, 1H, J=17.4 Hz), 5.12 (d, 1H, J=10.1 Hz), 4.74 (br, 4H), 4.47 (m,1H), 4.32 (br, 2H), 3.91 (m, 1H), 2.94 (m, 1H), 2.62 (m, 2H), 2.43 (m,1H), 2.24 (m, 2H), 2.1-1.70 (br, 3H), 1.45-1.10 (m, 22H).

MS m/z: 802.2 (M+Na⁺).

Starting from the Intermediate 1d, by coupling with(S,E)-2-(tert-butoxycarbonylamino)-7-methoxy-7-oxohept-5-enoic acid, thefollowing compound was prepared.

(3R,5S)-1-((S,E)-2-(tert-butoxycarbonylamino)-7-methoxy-7-oxohept-5-enoyl)-5-((1R,2S)-1-(cyclopropylsulfonylcarbamoyl)-2-vinylcyclopropylcarbamoyl)pyrrolidin-3-yl4-fluoroisoindoline-2-carboxylate

MS m/z: 798.3 (M+Na⁺).

Starting from the Intermediate 1d, the following compounds are preparedby coupling with the appropriate intermediates made similarly asdescribed in Step 6a:

(3R,5S)-1-(S)-2-(tert-butoxycarbonylamino)-5-oxooct-6-ynoyl)-5-((1R,2S)-1-(cyclopropylsulfonylcarbamoyl)-2-vinylcyclopropylcarbamoyl)pyrrolidin-3-yl4-fluoroisoindoline-2-carboxylate

(3R,5S)-1-((S)-2-(tert-butoxycarbonylamino)-3,3,7-trimethyl-5-oxooct-6-enoyl)-5-((1R,2S)-1-(cyclopropylsulfonylcarbamoyl)-2-vinylcyclopropylcarbamoyl)pyrrolidin-3-yl4-fluoroisoindoline-2-carboxylate

(3R,5S)-1-((S)-2-(tert-butoxycarbonylamino)-4,4,7-trimethyl-5-oxooct-6-enoyl)-5-((1R,2S)-1-(cyclopropylsulfonylcarbamoyl)-2-vinylcyclopropylcarbamoyl)pyrrolidin-3-yl4-fluoroisoindoline-2-carboxylate

(3R,5S)-1-((S)-2-(tert-butoxycarbonylamino)-6-methyl-4-oxohept-5-enoyl)-5-((1R,2S)-1-(cyclopropylsulfonylcarbamoyl)-2-vinylcyclopropylcarbamoyl)pyrrolidin-3-yl4-fluoroisoindoline-2-carboxylate

MS m/z: 760.1 (M+H⁺).

Starting from the Intermediate 1d, the following compound was preparedby coupling with the appropriate intermediates made similarly asdescribed in Step 6a:

(3R,5S)-1-(S)-2-(tert-butoxycarbonylamino)-8,8-dimethyl-5-oxonon-6-ynoyl)-5-((1R,2S)-1-(cyclopropylsulfonylcarbamoyl)-2-vinylcyclopropylcarbamoyl)pyrrolidin-3-yl4-fluoroisoindoline-2-carboxylate

MS m/z: 822.3 (M+Na⁺).

The following compound was made by palladium catalyzed hydrogenation ofIntermediate 1d, followed by coupling reaction described in step 6b:

(3R,5S)-1-(S)-2-(tert-butoxycarbonylamino)-7-methyl-5-oxooct-6-enoyl)-5-((1R,2R)-1-(cyclopropylsulfonylcarbamoyl)-2-ethylcyclopropylcarbamoyl)pyrrolidin-3-yl4-fluoroisoindoline-2-carboxylate

A total of 33 mg of the title compound was obtained from 100 mg ofIntermediate 1d: R_(t) 0.5 (EtOAc); MS m/z: 776.2 (M+H⁺).

In similar fashion, the following compounds are made:

(3R,5S)-1((S)-2-(tert-butoxycarbonylamino)-6-methyl-5-oxohept-6-enoyl)-5-((1R,2R)-1-(cyclopropylsulfonylcarbamoyl)-2-ethylcyclopropylcarbamoyl)pyrrolidin-3-yl4-fluoroisoindoline-2-carboxylate

(3R,5S)-1-((S)-2-(tert-butoxycarbonylamino)-5-cyclobutenyl-5-oxopentanoyl)-5-R,2R)-1-(cyclopropylsulfonylcarbamoyl)-2-ethylcyclopropylcarbamoyl)pyrrolidin-3-yl4-fluoroisoindoline-2-carboxylate

(3R,5S)-1-(S)-2-(tert-butoxycarbonylamino)-6-cyclobutylidene-5-oxohexanoyl)-5-((1R,2R)-1-(cyclopropylsulfonylcarbamoyl)-2-ethylcyclopropylcarbamoyl)pyrrolidin-3-yl4-fluoroisoindoline-2-carboxylate

(3R,5S)-1-((S,E)-2-(tert-butoxycarbonylamino)-8,8-dimethyl-5-oxonon-6-enoyl)-5-((1R,2R)-1-(cyclopropylsulfonylcarbamoyl)-2-ethylcyclopropylcarbamoyl)pyrrolidin-3-yl4-fluoroisoindoline-2-carboxylate

(3R,5S)-1-(S)-2-(tert-butoxycarbonylamino)-2-(S)-2-oxocyclopent-3-enyl)acetyl)-5-((1R,2R)-1-(cyclopropylsulfonylcarbamoyl)-2-ethylcyclopropylcarbamoyl)pyrrolidin-3-y4-fluoroisoindoline-2-carboxylate

In similar fashion, the following compound was made:

(3R,5S)-1-(S)-2-(tert-butoxycarbonylamino)-6-methyl-4-oxohept-5-enoyl)-5-((1R,2R)-1-(cyclopropylsulfonylcarbamoyl)-2-ethylcyclopropylcarbamoyl)pyrrolidin-3-yl4-fluoroisoindoline-2-carboxylate

MS m/z: 762.2 (M+Na⁺).

The following compound was made by prolonged palladium catalyzedhydrogenation (24-48 hours) of Intermediate 1d, followed by couplingreaction described in step 6b:

(3R,5S)-1-((S)-2-(tert-butoxycarbonylamino)-7-methyl-5-oxooct-6-enoyl)-5-(1-(cyclopropanesulfonamido)-1-oxohexan-2-ylcarbamoyl)pyrrolidin-3-yl4-fluoroisoindoline-2-carboxylate

A total of 58 mg of the title compound (I-44) was obtained from 100 mgof Intermediate 1d: R_(f) 0.5 (EtOAc); MS m/z: 800.2 (M+Na⁺).

The following scheme and procedure depict a synthesis of(S,E)-2-(tert-butoxycarbonylamino)-7-cyclo pentyl-5-oxohept-6-enoicacid:

To a stirring solution of 1 mmol diethyl methylphosphonate in 2 mL ofanhydrous THF under Ar at −78° C., was added dropwise 650 uL of 1.6 Mn-butyllithium solution in hexane. The resulting mixture was stirred 30min at −78° C. before N-Boc methylpyroglutamate (1 mmol) in 1 mL of THFwere added. The reaction mixture was then warmed slowly to roomtemperature, and stirred overnight. Aqueous NH₄Cl solution (5 mL) wasadded, and the reaction mixture was extracted with ethyl acetate (30mL). The organic layer was then dried over sodium sulfate, filtrated,and concentrated. The residue was purified by flash columnchromatography on silica gel with heptane/EtOAc 1/3 (v/v) as elutingsolvent, giving the phosphonate as a colorless oil 220 mg (55%).

To a solution of 105 mg of phosphonate from above (0.265 mmol), 52 mg ofcyclopentanyl carboxaldehyde (2 equiv.), 110 mg of potassium carbonatein 2 mL of THF and 2 mL of water was stirred vigorously overnight. Ethylacetate 30 mL was then added in, and the organic layer was dried oversodium sulfate. After concentration, the residue was purified by flashcolumn chromatography on silica gel with heptane/EtOAc 1/3 (v/v) aseluting solvent, giving a colorless oil (52 mg, 58%) as the desiredester.

The ester obtained was then subjected to basic hydrolysis in 1 mL of THFand 1 mL of t-butanol with 0.5 mL of 1 M aqueous LiOH solution. After 30min, 0.6 mL of 1 M HCl was added, and the resulting mixture wasextracted with 30 mL of ethyl acetate. After drying over anhydroussodium sulfate, the organic layer was filtrated, concentrated to givethe desired acid.

The following compound was prepared by coupling Intermediate 1d with(S,E)-2-(tert-butoxycarbonylamino)-7-cyclopentyl-5-oxohept-6-enoic acidmade above using HATU by the procedures described in previous examples:

(3R,5S)-1-((S,E)-2-(tert-butoxycarbonylamino)-7-cyclopentyl-5-oxohept-6-enoyl)-5-((1R,2S)-1-(cyclopropylsulfonylcarbamoyl)-2-vinylcyclopropylcarbamoyl)pyrrolidin-3-yl-4-fluoroisoindoline-2-carboxylate

MS m/z: 812.4 (ES−).

In similar manner, by using an appropriate aldehyde in the step for thesynthesis of(S,E)-2-(tert-butoxycarbonylamino)-7-cyclopentyl-5-oxohept-6-enoic aciddescribed above, the following compounds can be prepared:

Example 7

(1aR,3aS,5R,9S,16aS,Z)-11-(o-nitrophenylsulfonyl)-9-(tert-butoxycarbonylamino)-1a-(cyclopropylsulfonylcarbamoyl)-3,8-dioxo-1,1a,2,3,3a,4,5,6,8,9,10,11,12,13,14,16a-hexadecahydrocyclopropa[n]pyrrolo[2,1-c][1,4,8]-triazacyclopentadecin-5-yl-4-fluoroisoindoline-2-carboxylate(I-45): The title compound is prepared according to the steps andintermediates as described below:

Step 7a: Intermediate 7a

The Intermediate 7a was made following the procedure described for thesynthesis of Intermediate 3c by using 5-bromopent-1-ene as thealkylating reagent.

Step 7b: Intermediate 7b

The Intermediate 7b was made by coupling Intermediate 1d from Example 1and Intermediate 7a using HATU following the coupling reactionsdescribed for Intermediate 1e in Example 1. MS: 946.2 (M+1).

Step 7c: Compound I-45

To a solution of 540 mg of Intermediate 7b in 150 mL of anhydrousdichloromethane was added 100 mg1,3-Bis(2,4,6-trimethylphenyl)-4,5-dihydroimidazol-2-ylidene[2-(i-propoxy)-5-(N,N-dimethylaminosulfonyl)phenyl]methyleneruthenium(II) (Zhan catalyst 1B, RC-303, Zannan Pharma Ltd.) under nitrogen. Theresulting mixture was heated at 48° C. overnight. The LC-MS showedcomplete conversion to the product. Reaction solution was subject toflash column chromatography on silica gel with eluent (heptane/EtOAc v/v1:1 to pure EtOAc), giving 140 mg of the desired product. MS: 916.3(ES−).

In similar fashion, by starting with(S)-5-allylamino-2-tert-butoxycarbonylamino-pentanoic acid in step 7a,the following compound was prepared:

MS: 916.3 (ES−)

In similar fashion, by starting with(S)-4-[But-3-enyl-(2-nitro-benzenesulfonyl)-amino]-2-tert-butoxycarbonylamino-butyricacid in step 7a, the following compound was prepared:

MS: 916.3 (ES−)

Example 8

(1aR,3aS,5R,9S,16aS,Z)-11-acryloyl-9-(tert-butoxycarbonylamino)-1a-(cyclopropylsulfonylcarbamoyl)-3,8-dioxo-1,1a,2,3,3a,4,5,6,8,9,10,11,12,13,14,16a-hexadecahydrocyclopropa[n]pyrrolo[2,I-c][1,4,8]triazacyclopentadecin-5-yl-4-fluoroisoindoline-2-carboxylate(I-35)

The title compound was prepared according to the steps and intermediatesas described below:

Step 8a: Intermediate 8a:

The Intermediate 8a was made by treating compound I-45 from Example 7with thiophenol following the procedure described in step 3e. MS: 733.3(M+1).

Step 8b: Compound I-35:

The title compound was made by treating Intermediate 8a with acryloylchloride following the procedure described in step 3f. R_(f) 0.2 (5%MeOH in DCM); MS m/z: 787.3 (M+H⁺).

In similar fashion, by treating Intermediate 8a with chloroacetylchloride (1.2 eq), the following compound was prepared:

(1aR,3aS,5R,9S,16aS,Z)-11-chloroacetyl-9-(tert-butoxycarbonylamino)-1a-(cyclopropylsulfonylcarbamoyl)-3,8-dioxo-1,1a,2,3,3a,4,5,6,8,9,10,11,12,13,14,16a-hexadecahydrocyclopropa[n]pyrrolo[2,I-c][1,4,8]triazacyclopentadecin-5-yl-4-fluoroisoindoline-2-carboxylate(I-45)

MS: 831.2 (M+Na⁺).

In similar fashion, by treating Intermediate 8a with Chloroacetylchloride (3.0 eq), the following compound was prepared:

MS: 907.2 (M+Na⁺).

In similar fashion, by starting with compounds I-53, and I-54 in step8a, the following compounds were prepared:

(1aR,3aS,5R,9S,16aS,Z)-12-acryloyl-9-(tert-butoxycarbonylamino)-1a-(cyclopropylsulfonylcarbamoyl)-3,8-dioxo-1,1a,2,3,3a,4,5,6,8,9,10,11,12,13,14,16a-hexadecahydrocyclopropa[n]pyrrolo[2,I-c][1,4,9]triazacyclopentadecin-5-yl-4-fluoroisoindoline-2-carboxylate

MS: 785.3 (ES−).

(1aR,3aS,5R,9S,16aS,Z)-13-acryloyl-9-(tert-butoxycarbonylamino)-1a-(cyclopropylsulfonylcarbamoyl)-3,8-dioxo-1,1a,2,3,3a,4,5,6,8,9,10,11,12,13,14,16a-hexadecahydrocyclopropa[e]pyrrolo[1,2-a][1,4,10]triazacyclopentadecin-5-yl-4-fluoroisoindoline-2-carboxylate

MS: 787.3 (ES+), 785.3 (ES−).

By starting with compound I-67 and following the metathesis proceduredescribed in step 7c, the following compound was prepared.

(2R,6S,10E,12Z,13aS,14aR,16aS)-6-(tert-butoxycarbonylamino)-14a-(cyclopropylsulfonylcarbamoyl)-5,9,16-trioxo-2,3,5,6,7,9,13a,14,14a,15,16,16a-dodecahydro-1H-cyclopropa[i]pyrrolo[1,2-e][1,5,8]oxadiazacyclopentadecin-2-yl-4-fluoroisoindoline-2-carboxylate

MS: 744.2 (ES−).

Following the procedures described in Examples 7 and 8, by starting withthe appropriate intermediate in the place of intermediate 7a, thefollowing compound was prepared:

(2R,6S,13aS,14aR,16aS,Z)-9-((E)-but-2-enoyloxy)-6-(tert-butoxycarbonylamino)-14a-(cyclopropylsulfonylcarbamoyl)-5,16-dioxo-1,2,3,5,6,7,8,9,10,11,13a,14,14a,15,16,16a-hexadecahydrocyclopropa[e]pyrrolo[1,2-a][1,4]diazacyclopentadecin-2-yl4-fluoroisoindoline-2-carboxylate

MS: 838.2 (M+Na).

Example 9

The title compound is prepared according to the steps and intermediatesas described in the scheme below:

Methyl2,2,12,12-tetramethyl-4,9-dioxo-3,10-dioxa-5,8-diazahexadec-15-ene-7-carboxylate(Intermediate 9-1)

To a stirring solution of 381 mg of methyl2-amino-3-(tert-butoxycarbonylamino)propanoate hydrochloride, and 1 mLof Et₃N in 10 mL of anhydrous THF, was added 1.5 mmol of2,2-dimethylhex-5-enyl carbonochloridate. The resulting mixture wasstirred at rt overnight, then concentrated. The residue was subject tocolumn chromatography on silica gel with heptanes/EtOAc (v/v 7/1) aseluent, giving 500 mg colorless oil (90%). ¹HNMR (400 MHz, CDCl₃) d 5.82(m, 1H), 5.64 (br s, 1H), 5.02 (dd, 1H, J=13.2, 1.8 Hz), 4.95 (d, 1H,J=8.2 Hz), 4.83 (br s, 1H), 4.39 (br d, 1H, J=6.0 Hz), 3.83 (br s, 2H),3.80 (s, 3H), 2.55 (m, 2H), 2.02 (m, 2H), 1.43 (s, 9H), 1.35 (m, 2H),0.91 (s, 6H).

2,2,12,12-tetramethyl-4,9-dioxo-3,10-dioxa-5,8-diazahexadec-15-ene-7-carboxylicacid (Intermediate 9-2)

To a stirring mixture of 500 mg of intermediate 8-1 in a mixed solventof MeOH-THF (5 mL-5 mL), was added 420 mg of LiOH—H₂O followed by 5 mLof water. The reaction mixture was stirred at rt for 30 min, thenacidified using 12 mL of 1.0 N HCl, and extracted with 60 mL ofdichloromethane. The organic layer was washed with brine and dried overMgSO₄. After filtration and concentration, 460 mg of sticky oil wasobtained as desired product (95%). LC-MS: m/z=357.2 (ES−).

(2S,4R)-1-tert-butyl 2-methyl4-(4-bromoisoindoline-2-carbonyloxy)pyrrolidine-1,2-dicarboxylate(Intermediate 9-3)

To a solution containing 0.5 g of (2S,4R)-1-tert-butyl 2-methyl4-hydroxypyrrolidine-1,2-dicarboxylate (2 mmol) in 5 mL ofN,N-dimethylacetamide, was added 370 mg of carbonyl diimidazole (1.1equiv.). The reaction was heated at 60° C. for 1 hr, then 400 mg of4-bromoisoindole (2 mmol) was added. The reaction was continued at 60°C. overnight. After cooling down, the reaction mixture was extractedwith 50 mL of EtOAc, and washed with water, brine, and dried overNa₂SO₄. After filtration and concentration, the residue was purified byflash column chromatography on silica gel using heptanes/EtOAc (v/v5/2), giving white solid 745 mg (79%). LC-MS: m/z=369.2 (ES+, M+1-Boc).

(2S,4R)-1-tert-butyl 2-methyl4-(4-vinylisoindoline-2-carbonyloxy)pyrrolidine-1,2-dicarboxylate(Intermediate 9-4)

Under Ar, to a solution of 745 mg of intermediate 8-3 (1.58 mmol) in 30mL of de-gassed toluene, was added 170 mg of palladiumtetrakistriphenylphosphine followed by 700 uL of vinyl tributyltin (2.4mmol). The reaction mixture was heated at 100° C. overnight. Aftercooling down, the solvent was removed under reduced pressure, and theresidue was purified by flash column chromatography on silica gel withheptanes/EtOAc (v/v 9/1-3/1) as eluent, giving white solid 533 mg (81%).LC-MS: m/z=317.2 (ES+, M+1-Boc).

(2S,4R)-methyl1-((S)-3-(tert-butoxycarbonylamino)-2-((2,2-dimethylhex-5-enyloxy)carbonylamino)propanoyl)-4-(4-vinyl-2,3-dihydro-1H-indene-2-carbonyloxy)pyrrolidine-2-carboxylate(Intermediate 9-5)

Intermediate 9-4 was Boc-deprotected using 4 N HCl in dioxane asdescribed in Example 1. 288 mg of this dc-Boc intermediate (0.82 mmol)was added to a stirring mixture containing 358 mg of intermediate 9-2(1.0 mmol), 400 mg of HATU (1.05 mmol), 300 uL of DIPEA in 5 mL of THF.After overnight stirring, the reaction mixture was diluted with 60 mL ofEtOAc, and washed with saturated sodium bicarbonate, diluted HCl, anddried over Na₂SO₄. Concentration and purification by flash columnchromatography on silica gel with heptanes/EtOAc (1/1) giving whitesolid 485 mg (90%). LC-MS: m/z=557.3 (ES+, M+1-Boc).

Macrocyclic Intermediate 9-6

Under N₂, a diluted solution of 485 mg of intermediate 9-5 and 150 mgZhan's catalyst 1B were stirred in 100 mL of de-gassed dichloroethane at50° C. overnight. The reaction mixture was then passed through a shortcolumn, then eluted with heptanes/EtOAc (v/v 1/3). The concentratedfraction was then subject to pre-HPLC purification, giving 312 mg ofbrowny solid (70%). LC-MS: m/z=529.2 (ES+, M+1−Boc).

Macrocycle Intermediate 9-8

25 mg of intermediate 9-6 was hydrolyzed in 1 mL of MeOH and 1 mL of THFusing 1 mL of 1.0 N LiOH for 1 hr. 1.2 mL of 1.0 N HCl was then added,and the reaction mixture was extracted with 30 mL of dichloromethane.The organic layer was then washed with brine, and dried over MgSO₄.After filtration and concentration, the residue was redissolved in 1.5mL of anhydrous acetonitrile, 25 mg of HATU, 200 uL of DIPEA were thenadded followed by 13 mg of intermediate 9-7. After stirring at rt for 20min, the reaction mixture was concentrated and purified directly byprep-HPLC, giving 20 mg yellow solid (61%). LC-MS: m/z=727.2 (ES+,M+1-Boc, 825.2 (ES−).

I-40

To a solution of 20 mg of intermediate 9-8 stirred in 1 Ldichloromethane, was added 1.5 mL of 4.0 M HCl in dioxane. After 30 min,the reaction mixture was concentrated. To the residue was added 1.5 mLof acetonitrile, 200 uL of DIPEA, 20 mg of acrylic acid, and 50 mg ofHATU. After stirring at rt for 30 min, the reaction mixture wasconcentrated and purified by prep-HPLC, giving white solid 14.0 mg(68%). LC-MS: m/z=779.3 (ES−), 781.2 (ES+)

In similar fashion, by starting with Intermediate 3c instead ofIntermediate 3a, the following compound can be made:

In similar fashion, using the saturated form of Intermediate 9-6, thefollowing compound was made:

LC-MS: m/z=781.3 (ES−), 783.2 (ES+).

In similar fashion, if both Intermediate 9-7 and Intermediate 9-8 arehydrogenated before coupling, the following compound can be made:

Example 10

Enone-macrocyclic I-45

The title compound was prepared according to the steps and intermediatesas described in the scheme below:

(S)-2-benzyl 1-(2,2-dimethylhex-5-enyl)5-oxopyrrolidine-1,2-dicarboxylate (Intermediate 10-1)

At 0° C., to a stirring solution of 440 mg of5-Oxo-pyrrolidine-2-carboxylic acid benzyl ester (2 mmol), 300 uL oftriethylamine, 300 mg of N,N-dimethylaminopyridine (2.2 mmol) in 7 mL ofdichloromethane, was added 2 mmol of 4-pentenyl-1-yl chloroformate. Thereaction mixture was then warmed to rt, and stirred 24 hr. Afterconcentration, the resulting residue was dissolved in EtOAc 40 mL,washed with 6 mL of 1.0 N aq. HCl, brine, and dried over anhydroussodium sulfate. The organic solvent was evaporated under reducedpressure, and the residue was purified by flash chromatography on silicagel using heptane/EtOAc (v/v 4/1-2/1), giving 430 mg of colorless oil asintermediate 10-1 (58%).

2-((2,2-dimethylhex-5-enyloxy)carbonylamino)-7-methyl-5-oxooct-6-enoicacid (Intermediate 10-2)

The debenzylation was done with 13 mg of Pd(OAc)₂, 24 uL of Et₃N, 278 uLof Et₃SiH in 2 mL of dichloromethane at rt for 30 min. After filtration,the concentrated residue was subject to Grignard reagent (2.5 equiv)addition at −78° C. for 2 hr. After quenching with diluted acid, theproduct was extracted with dichloromethane and dried over MgSO₄. Theconcentrated product is desired intermediate 10-2, which was useddirectly for next step.

Following the General Procedures Described in Example 9

Intermediate 9-4 was de-Boced with 4 N HCl, then coupled withintermediate 10-2, which produced Intermediate 10-3. Intermediate 10-3underwent olefin metathesis using either Grubbs catalyst or Zhan'scatalyst, to give macrocycle Intermediate 10-4. After basic hydrolysiswith LiOH, the acid form of intermediate 10-4 was coupled withIntermediate 9-7, furnishing the title compound I-55. MS: m/e=806.3(ES⁻).

In similar fashion, compound I-56 was prepared when using the saturatedform of Intermediate 9-7:

MS m/z: 808.3 (ES−).

In similar fashion, compound I-79 was prepared when using (S)-benzyl4-oxoazetidine-2-carboxylate in the place of the5-Oxo-pyrrolidine-2-carboxylic acid benzyl ester in the first step:

¹HNMR (CD₃OD, 400 MHz) δ 8.80 (s, 1H), 7.12-7.25 (m, 3H), 6.36 (d, 1H,J=16.0 Hz), 6.15 (s, 1H), 6.04 (m, 1H), 5.75 (m, 1H), 5.35 (br t, 1H,J=4.0 Hz), 5.28 (d, 1H, J=2.0, 16.8 Hz), 5.10 (dd, 1H, J=2.0, 12.4 Hz),4.67 (m, 2H), 4.38 (dt, 2H, J=4.0, 11.2 Hz), 4.26 (d, 1H, J=11.6 Hz),4.12 (dd, 1H, J=3.6, 12.0 Hz), 3.40 (d, 1H, J=10.8 Hz), 3.11 (dd, 1H,J=7.2, 17.6 Hz), 2.82-2.92 (m, 2H), 2.60 (dd, 1H, J=6.8, 14.0 Hz),2.15-2.30 (m, 4H), 2.13 (s, 3H), 1.90 (s, 3H), 1.79 (dd, 1H, J=5.2, 8.0Hz), 1.40 (m, 3H), 1.20 (m, 2H), 1.06 (m, 1H), 0.99 (s, 3H), 0.88 (s,3H). MS: m/e=794.2 (ES).

Example 11 Single Chain HCV Protease (wt) Peptide Expression andPurification

The single-chain proteolytic domain (NS4A₂₁₋₃₂-GSGS-NS₃₃₋₆₃₁) was clonedinto pET-14b (Novagen, Madison, Wis.) and transformed into DH10B cells(Invitrogen). The resulting plasmid was transferred into Escherichiacoli BL21 (Novagen) for protein expression and purification as describedpreviously (1, 2). Briefly, the cultures were grown at 37° C. in LBmedium containing 100 μg/mL of ampicillin until the optical density at600 nm (OD600) reached 1.0 and were induced by addition ofisopropyl-β-D-thiogalactopyranoside (IPTG) to 1 mM. After an additionalincubation at 18° C. for 20 h, bacteria were harvested by centrifugationat 6,000×g for 10 min and resuspended in a lysis buffer containing 50 mMNa₃PO₄, pH 8.0, 300 mM NaCl, 5 mM 2-mercaptoethanol, 10% glycerol, 0.5%Igepal CA630, and a protease inhibitor cocktail consisting of 1 mMphenylmethylsulfonyl fluoride, 0.5 μg/mL leupeptin, pepstatin A, and 2mM benzamidine. Cells were lysed by freezing and thawing, followed bysonication. Cell debris was removed by centrifugation at 12,000×g for 30min. The supernatant was further clarified by passing through a 0.45-μmfilter (Corning) and then loaded onto a HiTrap chelating column chargedwith NiSO₄ (Amersham Pharmacia Biotech). The bound protein was elutedwith an imidazole solution in a 100- to-500 mM linear gradient. Selectedfractions were run through Ni²⁺ column chromatography and were analyzedon a 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel. The purifiedprotein was resolved by electrophoresis in a 12% SDS-PAGE gel and thentransferred onto a nitrocellulose membrane. The protein was analyzed byWestern blot analysis using monoclonal antibodies against NS3. Proteinswere visualized by using a chemiluminescence kit (Roche) withhorseradish peroxidase-conjugated goat anti-mouse antibodies (Pierce) assecondary antibodies. The protein was aliquoted and stored at −80° C.

Example 12 Cloning and expression of HCV protease A156S, A156T, D168A,D168V drug-resistance mutants and C159S variant

The mutant DNA fragments of NS4A/NS3 were generated by PCR and clonedinto pET expression vector. After transformation into BL21 competentcells, the expression was induced with IPTG for 2 hours. The His-taggedfusion proteins were purified using affinity column followed by sizeexclusion chromatography.

Example 13

Assay buffer: 2% CHAPS, 50 mM Tris pH 7.5, 50% glycerol, 2 uM M-2235(Bachcm) substrate. In a 50 ul reaction, add 49 ul assay buffer, 1 ul (1U) HCV serine protease (Bioenza). Incubate 20 minutes at roomtemperature. The plate was read at either 350/460 nm(excitation/emission) on a fluorescent micro-plate reader or monitoredat one-minute intervals to achieve the kinetic curve.

The enzyme tolerated 1% DMSO and 2% methanol. In the experiments oftesting compounds, the compounds in pure DMSO were diluted 10 times with20% methanol (10% DMSO and 20% methanol). This compound solution wasadded to the reaction (not exceeding 10% of the final reaction volume).The final concentration of the organic solvents was: 1% DMSO and 2%methanol.

Example 14 Additional Assay Protocols Method A:

The compounds were assayed to evaluate the antiviral activity andcytotoxicity of compounds in vitro using HCV RNA replicons. This assayused the cell line ET (luc-ubi-neo/ET), which is a human Huh7 hepatomacell line that contains an HCV RNA replicon with a stable luciferase(Luc) reporter and three cell culture-adaptive mutations. The HCV RNAlevels were directly measured by viral specific TaqMan RT-PCR:

Forward primer: (SEQ ID NO: 63) ACGCAGAAAGCGTCTAGCCAT Reverse primer:(SEQ ID NO: 64) TACTCACCGGTTCCGCAGA Probe: (SEQ ID NO: 65)[6-FAM]-CCTGGAGGCTGCACGACACTCAT-[TAMRA]

The ET cell line was grown in Dulbecco's modified essential media(DMEM), 10% fetal bovine serum (FBS), 1% penicillin-streptomycin(pen-strep), 1% glutamine, 250 μg/mL G418 in a 5% CO₂ incubator at 37°C. All cell culture reagents were obtained from Mediatech (Manassas,Va.). Cells were trypsinized (1% trypsin:EDTA) and plated out at 5×10³cells/well in white 96-well assay plates (Costar) dedicated to cellnumber (cytotoxicity) or antiviral activity assessments. Drugs wereadded at six 3-fold concentrations each and the assay was run in DMEM,5% FBS, 1% pen-strep, 1% glutamine. Human interferon alpha-2b (PBLBiolabs, New Brunswick, N.J.) was included in each run as a positivecontrol compound. Cells were processed 72 hr post drug addition when thecells are still subconfluent. Antiviral activity was measured byanalyzing replicon-derived luciferase activity using the Steady-GloLuciferase Assay System (Promega, Madison, Wis.) according tomanufacturer's instruction. The number of cells in each well wasdetermined by CytoTox-1 reagent (Promega). Compound profile was derivedby calculating applicable EC₅₀ (effective concentration inhibiting virusreplication by 50%), EC₉₀ (effective concentration inhibiting virusreplication by 90%), IC₅₀ (concentration decreasing cell viability by50%) and SI₅₀ (selective index: EC₅₀/IC₅₀) values. IC₅₀ values forselected compounds are set forth in Table 5, below.

Method B: HCV Protease Assay Using Fret Methodology

A quantitative, fluorescence resonance energy transfer (FRET)-basedmethodology was employed to identify HCV NS3/4A protease inhibitors. Theassay employed a synthetic FRET peptide, derived from the HCV NS5A/5Bcleavage site, with the HCV protease to evaluate the activity ofcompounds against the protease by monitoring the cleavage activity ofthe complex. A synthetic peptide which encompasses the NS5A-5B junction(NH2-EDVVCCSMSYK-COOH) was labeled with Dabcyl and Edans at N- andC-termini, respectively (Invitrogen, Carlsbad, Calif.). Fluorescencemeasurement was used to estimate the IC₅₀ value of the test compound.The two fluorophores form a quenching pair and exhibit FRET within theintact peptide. Upon cleavage of the FRET peptide by HCV NS3/4Aproteinase complex (100 ng/mL), the fluorescence is recovered and can becontinuously monitored at excitation/emission=340/490 nm.

Example 15

HCV Protease FRET Assay for Wild Type and Mutated NS3/4A 1b Enzymes(IC₅₀)

The following protocol was used to generate IC₅₀ values as depicted forcompound 239 in Table 4, above. The protocol is a modified FRET-basedassay (v_(—)02) from In Vitro Resistance Studies of HCV Serine ProteaseInhibitors, 2004, JBC, vol. 279, No. 17, pp 17508-17514. Inherentpotency of compounds was assessed against A156S, A156T, D168A, and D168Vmutants of the HCV NS3/4A 1b protease enzyme as follows:

10× stocks of NS3/4A protease enzyme from Bioenza (Mountain View,Calif.) and 1.13×5-FAM/QXL™520 FRET peptide substrate from Anaspec (SanJose, Calif.) were prepared in 50 mM HEPES, pH 7.8, 100 mM NaCl, 5 mMDTT and 20% glycerol. 5 μL of each enzyme were pre-incubated in aCorning (#3573) 384-well, black, non-treated microtiter plate (Corning,N.Y.) for 30 min at 25° C. with a 0.5 μL volume of 50% DMSO and seriallydiluted compounds prepared in 50% DMSO. Protease reactions were startedwith the addition of 45 μL of the FRET substrate and monitored for 120minutes at λ_(ex)487/λ_(em)514 through Quad⁴ monochromoters in aSynergy⁴ plate reader from BioTek (Winooski, Vt.). At the conclusion ofeach assay, progress curves from each well were examined for linearreaction kinetics and fit statistics (R², absolute sum of squares).Initial velocity (0 minutes to 30+ minutes) from each reaction wasdetermined from the slope of a plot of relative fluorescence units vstime (minutes) and then plotted against inhibitor concentration toestimate IC₅₀ from log [Inhibitor] vs Response, Variable Slope model inGraphPad Prism from GraphPad Software (San Diego, Calif.). IC₅₀ valuesfor selected compounds are set forth in Table 5, below.

Table 5 shows the activity of selected compounds of this invention inthe FRET Assay. The compound numbers correspond to the compound numbersin Table 3. Compounds having an activity designated as “A” provided anIC₅₀≦10 nM; compounds having an activity designated as “B” provided anIC₅₀ 10-100 nM; compounds having an activity designated as “C” providedan IC₅₀ of 100-1000 nM; compounds having an activity designated as “D”provided an IC₅₀ of 1000-10,000 nM; and compounds having an activitydesignated as “E” provided an IC₅₀≧10,000 nM.

TABLE 5 Enzymatic Data for Exemplary Compounds (IC₅₀) Compound testedEnzyme/Assay Inhibition (I-1) WT A HCV A156S A HCV A156T A HCV D168A AHCV D168V A (I-6) WT A HCV A156S A HCV A156T A HCV D168A B HCV D168V B(I-7) WT B D168A D (I-11) WT A D168A B

Example 16 HCV Protease FRET Assay for WT and Mutated NS3/4A 1b Enzymes(IC₅₀ _(—) _(APP))

The following protocol was used to generate “apparent” IC₅₀(IC_(50 APP)) values as depicted in Table 6, below. Without wishing tobe bound by any particular theory, it is believed that IC_(50di —)_(APP), contrasted with IC₅₀ values, may provide a more usefulindication of time-dependent inhibition, and are thus morerepresentative of binding affinity. The protocol is a modifiedFRET-based assay (v_(—)03) developed to evaluate compound potency,rank-order and resistance profiles against wild type and C159S, A156S,A156T, D168A, D168V, R155K mutants of the HCV NS3/4A 1b protease enzymeas follows: 10× stocks of NS3/4A protease enzyme from Bioenza (MountainView, Calif.) and 1.13×5-FAM/QXL™520 FRET peptide substrate from Anaspec(San Jose, Calif.) were prepared in 50 mM Tris-HCl, pH 7.5, 5 mM DTT, 2%CHAPS and 20% glycerol. 5 μL of each enzyme were added to Corning(#3575) 384-well, black, microtiter plates (Corning, N.Y.) afterspotting a 0.5 μL volume of 50% DMSO and serially diluted compoundsprepared in 50% DMSO. Protease reactions were immediately started afterenzyme addition with the addition of 45 μL of the FRET substrate andmonitored for 60-90 minutes at λ_(ex)485/λ_(em)520 in a Synergy⁴ platereader from BioTek (Winooski, Vt.). At the conclusion of each assay,progress curves from each well were examined for linear reactionkinetics and fit statistics (R², 95% confidence intervals, absolute sumof squares). Initial velocity (0 minutes to 15+ minutes) from eachreaction was determined from the slope of a plot of relativefluorescence units vs time (minutes) and then plotted against inhibitorconcentration as a percent of the no inhibitor and no enzyme controls toestimate apparent IC₅₀ from log [Inhibitor] vs Response, Variable Slopemodel in GraphPad Prism from GraphPad Software (San Diego, Calif.).

Table 6 shows the activity of selected compounds of this invention inthe FRET Assay. The compound numbers correspond to the compound numbersin Table 3. Compounds having an activity designated as “A” provided anIC₅₀≦10 nM; compounds having an activity designated as “B” provided anIC₅₀>10 nM and ≦100 nM; compounds having an activity designated as “C”provided an IC₅₀>100 nM and ≦1000 nM; compounds having an activitydesignated as “D” provided an IC₅₀>1000 nM and <10,000 nM; and compoundshaving an activity designated as “E” provided an IC₅₀≧10,000 nM.

TABLE 6 Enzymatic Data for Exemplary Compounds Compound testedEnzyme/Assay Inhibition (I-1) WT A HCV A156S A HCV A156T B HCV D168A CHCV D168V B Replicon ² B Rcplicon ² B ¹ (I-2) WT A HCV A156S A HCV A156TA HCV D168A A HCV D168V A Replicon ² B Replicon ² C ¹ (I-5) WT A HCVA156S A HCV A156T B HCV D168A C HCV D168V B Replicon ² B Replicon ² C ¹(I-6) WT A HCV A156S A HCV A156T A HCV D168A C HCV D168V C Replicon ² CReplicon ² D ¹ (I-7) WT C HCV A156S C HCV A156T C HCV D168A D HCV D168VD (I-8) WT A HCV A156S A HCV A156T A HCV D168A A HCV D168V B (I-9) WT AHCV A156S A HCV A156T A HCV D168A B HCV D168V C (I-10) WT C HCV A156S CHCV A156T D HCV D168A D HCV D168V D (I-11) WT A HCV A156S A HCV A156T AHCV D168A B HCV D168V B HCV R155K B Replicon ² A Replicon ² B ¹ (I-13)WT A HCV A156S A HCV A156T A HCV D168A C HCV D168V B Replicon ² BReplicon ² C ¹ (I-19) WT A HCV A156S A HCV A156T A HCV D168A B HCV D168VB (I-20) WT A HCV A156S A HCV A156T A HCV D168A B HCV D168V B Replicon ²B Replicon ² C ¹ (I-22) WT A HCV A156S A HCV A156T B HCV D168A C HCVD168V C HCV R155K B Replicon ² B Replicon ² B ¹ (I-23) WT A HCV A156S AHCV A156T A HCV D168A A HCV D168V B Replicon ² B Replicon ² C ¹ (I-24)WT A HCV A156S A HCV A156T A HCV D168A A HCV D168V A Replicon ² BReplicon ² C ¹ (I-25) WT A HCV A156S A HCV A156T B HCV D168A C HCV D168VC HCV R155K B Replicon ² A Replicon ² B ¹ (I-26) WT A HCV A156S A HCVA156T A HCV D168A A HCV D168V A (I-27) WT A HCV A156S A HCV A156T A HCVD168A A HCV D168V A Replicon ² B Replicon ² C ¹ (I-29) WT A HCV A156S AHCV D168A A HCV R155K A Replicon B ³ (I-32) WT A HCV A156S A HCV D168A BHCV R155K B HCV C159S A HCV D168V B HCV A156T A Replicon A ³ (I-33) WT AHCV A156S B HCV D168A D HCV R155K C (I-35) WT A HCV A156S A HCV D168A BHCV R155K B Replicon C ³ Replicon C ⁴ (I-36) WT A HCV A156S A HCV D168AA HCV R155K A Replicon C ³ Replicon C ⁴ (I-37) WT A HCV A156S A HCVD168A B HCV R155K B Replicon C ³ Replicon D ⁴ (I-39) WT A HCV A156S AHCV D168A C HCV R155K B (I-40) WT A HCV A156S A HCV D168A B HCV R155K A(I-42) WT A HCV A156S A HCV D168A B HCV R155K B (I-44) WT A HCV A156S BHCV D168A D HCV R155K D (I-45) WT A HCV A156S A HCV D168A A HCV R155K AReplicon B ³ Replicon C ⁴ (I-46) WT A HCV A156S A HCV D168A B HCV R155KA Replicon D ³ Replicon D ⁴ (I-47) WT A HCV A156S A HCV D168A B HCVR155K B Replicon B ³ Replicon C ⁴ (I-48) WT B HCV A156S B HCV D168A CHCV R155K C (I-49) WT C HCV A156S C HCV D168A D HCV R155K D Replicon D ³Replicon D ⁴ (I-50) WT B HCV A156S C HCV D168A D HCV R155K D Replicon D³ Replicon D ⁴ (I-51) WT A HCV A156S A HCV D168A B HCV R155K B RepliconC ³ Replicon D ⁴ (I-52) WT B HCV D168A D Replicon D ³ Replicon D ⁴(I-53) WT A HCV A156S A HCV D168A A HCV R155K A Replicon B ³ Replicon C⁴ (I-54) WT A HCV A156S A HCV D168A B HCV R155K B Replicon C ³ RepliconE ⁴ (I-55) WT A HCV A156S A HCV A156T A HCV D168A B HCV D168V C HCVR155K B Replicon A ³ (I-56) WT A HCV A156S A HCV D168A C HCV D168V D HCVR155K C Replicon A ³ (I-60) WT A HCV A156S B HCV D168V C HCV R155K C(I-65) WT A HCV A156S A HCV D168A C HCV R155K B (I-66) WT A HCV A156S AHCV D168A A HCV R155K A (I-67) WT A HCV A156S B HCV D168A C HCV R155K C(I-68) WT A HCV A156S A HCV D168A A HCV R155K A Replicon C ³ (I-69) WT AHCV A156S A HCV D168A B HCV R155K A Replicon C ³ (I-70) WT A HCV A156S AHCV D168A C HCV R155K C (I-71) WT A HCV A156S A HCV D168A C HCV R155K CReplicon A ³ (I-72) WT A HCV A156S A HCV D168A C HCV R155K B (I-79) WT AHCV A156S A HCV D168A B HCV R155K A Replicon A ³ ¹ Designates IC₉₀ value(nM). ² Data collected from assay described in Example 14. ³ DesignatesEC₅₀ value (nM). Data collected from assay described in Example 26. ⁴Designates EC₉₀ value (nM). Data collected from assay described inExample 26.

Example 17

Mass spectrometric analysis of HCV wild type or HCV variant C159S in thepresence of test compound is performed. 100 pmols of HCV wild type(Bioenza CA) is incubated with test compound for 1 hr and 3 hrs at10-fold access of test compound to protein. 1 ul aliquots of the samples(total volume of 4.24 ul) are diluted with 1.0 ul of 0.1% TFA prior tomicro C4 ZipTipping directly onto the MALDI target using Sinapinic acidas the desorption matrix (10 mg/mL in 0.1% TFA:Acetonitrile 50:50).Analyses are performed on a Shimadzu Biotech Axima TOF² (ShimadzuInstruments) matrix-assisted-laser desorption/ionization Time-of-Flight(MALDI-TOF) mass spectrometer. The same procedure is carried out on 100pmols of HCV C159S mutant of HCV protease for 3 hrs at 10-fold excess oftest compound to protein.

Example 18 Modification of Cys159 of Wild-Type HCV Protease Using aTryptic Digest Strategy

HCV is incubated with test compound for 3 hrs prior to trypticdigestion. Iodoacetamide is used as the alkylating agent after compoundincubation. For tryptic digests a 2 ul aliquot (0.06 ug/ul) is dilutedwith 10 ul of 0.1% TFA prior to micro C18 Zip Tipping directly onto theMALDI target using alpha cyano-4-hydroxy cinnamic acid as the matrix (5mg/mL in 0.1% TFA:Acetonitrile 50:50).

For tryptic digests the instrument is set in Reflectron mode with apulsed extraction setting of 1800. Calibration is done using the LaserBiolabs Pep Mix standard (1046.54, 1296.69, 1672.92, 2093.09, 2465.20).For CID/PSD analysis the peptide is selected using cursors to set iongate timing and fragmentation occurred at a laser power about 20% higherand He is used as the collision gas for CID. Calibration for fragmentsis done using the P14R fragmentation calibration for the Curved fieldReflectron.

Example 19

As depicted in FIGS. 1 and 2, mass spectrometric analysis of HCV wildtype in the presence of test compounds I-1 and I-25 was performed usingthe following protocol: HCV NS3/4A wild type (wt) was incubated for 1 hrat a 10× fold access of test compound to protein. 2 ul aliquots of thesamples were diluted with 10 ul of 0.1% TFA prior to micro C4 ZipTippingdirectly onto the MALDI target using Sinapinic acid as the desorptionmatrix (10 mg/ml in 0.1% TFA:Acetonitrile 50:50). For intact proteinmass measurement the instrument was set in linear mode using a pulsedextraction setting of 24,500 and apomyoglobin as the standard tocalibrate the instrument.

As depicted in FIG. 1, compared to the protein with no compound, theprotein incubated with compound I-1 has reacted significantly to producea new species at MW 25,218 Da, which is approximately 751 Da heavier andconsistent with the mass of compound I-1 at 747 Da.

As depicted in FIG. 2, after 1 hour reaction time there was conversionto a new peak at MH+ of 25,240 Da which is 773 Da heavier and consistentwith the mass of compound I-25.

Compounds I-2, I-5, I-6, I-8, I-9, I-10, I-13, I-19, I-20, I-22, I-23,I-24, I-26, and I-27 were tested in a similar fashion, using the methodsdescribed in Example 17, and measurable covalent modification of HCVNS3/4A wild type was observed.

Example 20

As depicted in FIGS. 4, 5, 6, and 7, mass spectrometric analysis of HCVmutants in the presence of compound I-11 was performed. HCV Mutants(A156S), (R155K), (D168A), (A156T), and (D168V) were incubated for 3 hrsat a 10× fold access of test compound to protein. 2 ul aliquots of thesamples were diluted with 10 ul of 0.1% TFA prior to micro C4 ZipTippingdirectly onto the MALDI target using Sinapinic acid as the desorptionmatrix (10 mg/ml in 0.1% TFA:Acetonitrile 50:50). For intact proteinmass measurement the instrument was set in linear mode using a pulsedextraction setting of 24,500 and apomyoglobin as the standard tocalibrate the instrument.

As depicted in FIG. 4, for the HCV (D168V) mutant there is completeconversion after 3 hours reaction time. The mass difference between thenew species and the unreacted mutant is consistent with the mass ofcompound I-11.

As depicted in FIG. 5, for the HCV (A156S) mutant there is completeconversion after 3 hours reaction time. The mass difference between thenew species and the unreacted mutant is consistent with the mass ofcompound I-11.

As depicted in FIG. 6, for the HCV (R155K) mutant there is goodconversion after 3 hours reaction time. The mass difference between thenew species and the unreacted mutant is consistent with the mass ofcompound I-11.

As depicted in FIG. 7, for the HCV (A156T) mutant there is completeconversion after 3 hours reaction time. The mass difference between thenew species and the unreacted mutant is consistent with the mass ofcompound I-11.

Compounds I-19 and I-24 were tested in a similar fashion using themethods described in Example 18, and measurable covalent modification ofHCV NS3/4A D168A was observed.

Example 21 Cell Culture

Huh-luc/neo-ET, Huh7-Lunet were obtained from ReBLikon Gmbh (Heidelberg,Germany). Cells were grown in Dulbecco modified Eagle medium (DMEM;Invitrogen) supplemented with 2 mM L-glutamine, nonessential aminoacids, 100 U of penicillin/ml, 100 μg of streptomycin/mL, and 10% fetalbovine serum. G418 (Geneticin; Invitrogen) was added at a finalconcentration of 400 ug//mL. Huh7-Lunet were grown in the absence ofG418.

Example 22 Mutant Constructs

Constructs containing clinically relevant mutations were generated byperforming site-directed mutagenesis on thepFK-1389-luc-ubi-neo-NS3-3′ET plasmid (ReBLikon Gmbh (Heidelberg,Germany)). using the QuickChange II Site-Directed Mutagenesis Kit(Stratagene, La Jolla, Calif.) according to manufacturer's directionsand with the primers described in Table 7, below.

TABLE 7 Primer sequence used to establish Mutant Replicon cell lines.NS3-A156S-F GCTGTGGGCATCTTTCGGTCTGCCGTGTGC SEQ ID NO: 66 ACCCGAGGGNS3-A156S-R CCCTCGGGTGCACACGGCAGACCGAAAGATGCCC SEQ ID NO: 67 ACAGCNS3-A156T-F GCTGTGGGCATCTTTCGGACTGCCGTGTGCACCC SEQ ID NO: 68 GAGGGNS3-A156T-R CCCTCGGGTGCACACGGCAGTCCGAAAGATGCCC SEQ ID NO: 69 ACAGCNS3-D168A-F GGGGTTGCGAAGGCGGTGGCCTTTGTACCCGTCG SEQ ID NO: 70 AGTCTNS3-D168A-R AGACTCGACGGGTACAAAGGCCACCGCCTTCGCA SEQ ID NO: 71 ACCCCNS3-D168V-F GGGGTTGCGAAGGCGGTGGTCTTTGTACCCGTCG SEQ ID NO: 72 AGTCTNS3-D168V-R AGACTCGACGGGTACAAAGACCACCGCCTTCGCA SEQ ID NO: 73 ACCCCNS3-C159S-F ATCTTTCGGGCTGCCGTGAGCACCCGAGGGGTTG SEQ ID NO: 74 CGAAGNS3-C159S-R CTTCGCAACCCCTCGGGTGCTCACGGCAGCCCGA SEQ ID NO: 75 AAGATNS3-R155K-F CACGCTGTGGGCATCTTTAAGGCTGCCGTGTGCA SEQ ID NO: 76 CCCGANS3-R155K-R TCGGGTGCACACGGCAGCCTTAAAGATGCCCACA SEQ ID NO: 77 GCGTG

Example 23 In Vitro Transcription

In vitro transcripts of HCV positive strands were generated by using theprotocol described by Lohmann V et al., J. Virol., 77:3007-3019, 2003.For transcription of positive-strand HCV RNAs, plasmid DNA (pFK 1341PI-Luc/NS3-3′/ET, obtained from ReBLikon Gmbh (Heidelberg, Germany)),was digested with Asel followed by Scat . After restriction digest, DNAwas extracted with phenol and chloroform, precipitated with ethanol, anddissolved in RNase-free water. In vitro transcription reactionscontained 80 mM HEPES (pH 7.5), 12 mM MgCl₂, 2 mM spermidine, 40 mMdithiothreitol, a 3.125 mM concentration of each nucleosidetriphosphate, 1 U of RNasin. 5 ug of restricted plasmid DNA and 80 U ofT7 RNA polymerase (Promega) was used. After 2 h at 37° C., an additional40 U of T7 polymerase was added, and the reaction was incubated foranother 2 h. Transcription was terminated by the addition of 1 U ofRNase-free DNase (Promega) per ug of plasmid DNA, followed by incubationfor 30 min at 37° C. After extraction with acidic phenol and chloroform,RNA was precipitated with isopropanol and dissolved in RNase-free water.The concentration was determined by measurement of the optical densityat 260 nm (OD260), and RNA integrity was checked by denaturing agarosegel electrophoresis.

Example 24 Transfection of HCV Full Length Genome and Selection ofStable Cell Lines

7×10⁴ Huh7-Lunet cells were seeded over night in a 12 well plate, thenext day 1 ug of RNA/well was transfected using Minis Tx (Madison, Wis.)kit. Transfection was performed according to manufacturer'sinstructions, and 24 hours after transfection cells were eithersubjected to Luciferase assay or subjected to G418 (400 ug/ml) selectionin order to establish stable cell lines.

Example 25 Inhibition of Protease Self Cleavage

Huh-7-Luc-Neo-ET cells were plated in Replicon Assay Medium (RPMIsupplemented with 5% FBS, 1× non-essential amino acids and pen/strep) ata density of 1×10⁵ cells/well in 12 well plates. Eight hours later themedia was removed and replaced with 1 ml media containing test compound(5 wells per compound) and 0.02% DMSO and the cells were returned to theincubator overnight. Sixteen hours later 1 well from each compound and 1untreated well were washed with PBS, then lysed and scraped into 30 ulof Cell Extraction Buffer (Biosource, Camarillo, Calif.) plus CompleteProtease Inhibitor (Roche, Indianapolis, Ind.). The remaining wells wererinsed 2× with PBS then fed with Replicon Media and returned to theincubator. Cells were washed once every hour by removing the old mediaand replacing it with fresh media and were lysed and collected at 4, 12,24, and 48 hours following the first collection.

Cell lysates were separated by SDS-Page (4-20%) and transferred toImmobilon-P PVDF membrane (Millipore Corporation, MA) and blotted withpolyclonal anti NS3 antibody (Bioenza, CA). Blots were scanned on anOdyssey infrared scanner from Licor and the FL band and cleavageproducts were quantified separately using the Licor software providedwith the scanner. The cleavage product was calculated as a percentage ofthe total NS3 in each sample and then normalized to the DMSO control sothat the DMSO control reflects 100% activity.

Results and Discussion

When protease activity is inhibited, self-cleavage is abolished and theonly protein species detectable is the holoenzyme. After 16 hours ofcontinuous exposure of the replicon cells to NS3 inhibitor compound, theself-cleavage products were undetectable in the treated samples, butreadily detectable in the not treated control replicon cells. Prolongedduration of action was demonstrated by exposing the replicon cells to aprotease inhibitor for 16 hours, at which time the compound was removed,and the replicon cells were repeatedly washed for several more hours.Covalent irreversible NS3 inhibitors demonstrated sustained inhibitionof NS3 internal self-cleavage activity for up to 48 hours, whereas theprotease self-cleavage activity rapidly returned when using reversiblecompounds (FIG. 8).

FIG. 8 depicts that the NS3 internal self-cleavage products areinhibited by treatment of replicon cells with Compound I-47 for 16hours.

FIGS. 9 and 9-A depict an irreversible covalent inhibitor (compoundI-11) of NS3 protease demonstrate prolonged inhibition of NS3 proteaseactivity in the wild-type replicon cells, as measured by self-cleavage,after the compounds are removed. The compound was incubated withreplicon cells for 16 hours and then removed (time 0). Even up to 48hours after removal of a covalent irreversible NS3 inhibitor, NS3self-cleaving activity is inhibited by at least 50%, whereas areversible drug, VX-950, shows virtually complete return of activity inas little as 4 hours after drug removal.

FIG. 10 depicts an irreversible covalent inhibitor (compound I-25) ofNS3 protease demonstrate prolonged inhibition of NS3 protease activityin the wild-type replicon cells, as measured by self-cleavage, after thecompounds are removed. The compound was incubated with replicon cellsfor 16 hours and then removed (time 0). Even up to 48 hours afterremoval of a covalent irreversible NS3 inhibitor, NS3 self-cleavingactivity is inhibited by at least 40%, whereas a reversible drug,VX-950, shows virtually complete return of activity in as little as 8hours after drug removal.

Example 26 Luciferase Assay

The compounds were assayed to evaluate the antiviral activity andcytotoxicity of compounds using replicon-derived luciferase activity.This assay used the cell line ET (luc-ubi-neo/ET), which is a human Huh7hepatoma cell line that contains an HCV RNA replicon with a stableluciferase (Luc) reporter and cell culture-adaptive mutations. The ETcell line was grown in a 5% CO₂ incubator at 37° C. in Dulbccco'smodified essential media (DMEM) supplemented with 2 mM L-glutamine,nonessential amino acids, 100 U of penicillin/ml, 100 μg ofstreptomycin/mL, and 10% fetal bovine serum. G418 (Geneticin;Invitrogen) was added at a final concentration of 400 ug//mL.

All cell culture reagents were obtained from Invitrogen (Carlsbad).Cells were trypsinized (1% trypsin:EDTA) and plated out at 5×10³cells/well in white 96-well assay plates (Costar) dedicated to cellnumber (cytotoxicity) or antiviral activity assessments. Test compoundswere added at six 3-fold concentrations each and the assay was run inDMEM, 5% FBS, 1% pen-strep, 1% glutamine, 1% non essential amino acid.Human interferon alpha-2b (PBL Biolabs, New Brunswick, N.J.) wasincluded in each run as a positive control compound. Cells wereprocessed 72 hr post test compound addition when the cells were stillsubconfluent. Antiviral activity was measured by analyzingreplicon-derived luciferase activity using the Steady-Glo LuciferaseAssay System (Promega, Madison, Wis.) according to manufacturer'sinstruction. The number of cells in each well was determined by CellTiter Blue Assay (Promega). Compound profile was derived by calculatingapplicable EC₅₀ (effective concentration inhibiting virus replication by50%), EC₉₀ (effective concentration inhibiting virus replication by90%), IC₅₀ (concentration decreasing cell viability by 50%) and SI₅₀(selective index: EC₅₀/IC₅₀) values.

While we have described a number of embodiments of this invention, it isapparent that our basic examples may be altered to provide otherembodiments that utilize the compounds and methods of this invention.Therefore, it will be appreciated that the scope of this invention is tobe defined by the appended claims rather than by the specificembodiments that have been represented by way of example.

1-24. (canceled)
 25. A method for inhibiting HCV protease, or a mutantthereof, in a patient comprising the step of administering to a patientin need thereof a compound of formula I:

or a pharmaceutically acceptable salt thereof, wherein: R¹ and R^(1′)are independently hydrogen or optionally substituted C₁₋₆ aliphatic, orR¹ and R^(1′) are taken together to form an optionally substituted 3-7membered carbocyclic ring; R^(2a) is —OH or —NHSO₇R²; R² is —N(R)₂ or anoptionally substituted group selected from C₃₋₇ cycloalkyl, a bridgedbicyclic, 6-10 membered aryl, 5-10 membered heteroaryl having 1-4heteroatoms independently selected from nitrogen, oxygen, or sulfur, or4-7 membered heterocyclyl having 1-2 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur; each R is independently hydrogen,optionally substituted C₁₋₆ aliphatic, or: two R on the same nitrogenatom are taken together with the nitrogen to form a 4-7 memberedheterocyclic ring having 1-2 heteroatoms independently selected fromnitrogen, oxygen, or sulfur; R³ is selected from the group consistingof:

R⁴ is H, —NHC(O)R⁵, —NHC(O)OR⁶,

or a natural or unnatural amino acid side-chain group; each R⁵ isindependently —N(R)₂ or an optionally substituted group selected fromC₁₋₆ aliphatic, a bridged bicyclic, 6-10 membered aryl, 5-10 memberedheteroaryl having 1-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur, or 4-7 membered heterocyclyl having 1-2 heteroatomsindependently selected from nitrogen, oxygen, or sulfur; R⁶ is anoptionally substituted group selected from C₁₋₆ aliphatic, a bridgedbicyclic, 6-10 membered aryl, 5-10 membered heteroaryl having 1-4heteroatoms independently selected from nitrogen, oxygen, or sulfur, or4-7 membered heterocyclyl having 1-2 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur; and R⁷ is an optionally substitutedgroup selected from C₁₋₆ aliphatic, a bridged bicyclic, 6-10 memberedaryl, 5-10 membered heteroaryl having 1-4 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur, or 4-7 membered heterocyclylhaving 1-2 heteroatoms independently selected from nitrogen, oxygen, orsulfur; and R^(z) is

or R⁴ and R^(z) are taken together with their intervening atoms to forman optionally substituted, saturated or unsaturated 16-22 membered ringhaving 2-6 heteroatoms independently selected from nitrogen, oxygen, orsulfur; each occurrence of R^(y) is independently selected from halogen,—OR^(◯), —CN, —NO₂, —N(R^(◯))₂, or optionally substituted C₁₋₄aliphatic; R^(◯) is an optionally substituted group selected from C₁₋₆aliphatic; m is an integer from 0 to 4, inclusive; s is an integer from0 to 4, inclusive; t is an integer from 0 to 4, inclusive; wherein thesum of s and t is non-zero.
 26. The method according to claim 25,wherein the HCV protease, or a mutant thereof, activity is inhibitedirreversibly.
 27. The method according to claim 25, wherein the HCVprotease, or a mutant thereof, activity is inhibited irreversibly bycovalently modifying Cys159 of HCV protease subtype 1b.
 28. The methodaccording to claim 25, wherein the HCV protease, or a mutant thereof,activity is inhibited irreversibly by covalently modifying Cys16.
 29. Amethod for treating an HCV protease-mediated disorder in a patient,comprising the step of administering to said patient a compound offormula I:

or a pharmaceutically acceptable salt thereof, wherein: R¹ and R^(1′)are independently hydrogen or optionally substituted C₁₋₆ aliphatic, orR¹ and R^(1′) are taken together to form an optionally substituted 3-7membered carbocyclic ring; R^(2a) is —OH or —NHSO₂R²; R² is —N(R)₂ or anoptionally substituted group selected from C₃₋₇ cycloalkyl, a bridgedbicyclic, 6-10 membered aryl, 5-10 membered heteroaryl having 1-4heteroatoms independently selected from nitrogen, oxygen, or sulfur, or4-7 membered heterocyclyl having 1-2 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur; each R is independently hydrogen,optionally substituted C₁₋₆ aliphatic, or: two R on the same nitrogenatom are taken together with the nitrogen to form a 4-7 memberedheterocyclic ring having 1-2 heteroatoms independently selected fromnitrogen, oxygen, or sulfur; R³ is selected from the group consistingof:

R⁴ is H, —NHC(O)R⁵, —NHC(O)OR⁶,

or a natural or unnatural amino acid side-chain group; each R⁵ isindependently —N(R)₂ or an optionally substituted group selected fromC₁₋₆ aliphatic, a bridged bicyclic, 6-10 membered aryl, 5-10 memberedheteroaryl having 1-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur, or 4-7 membered heterocyclyl having 1-2 heteroatomsindependently selected from nitrogen, oxygen, or sulfur; R⁶ is anoptionally substituted group selected from C₁₋₆ aliphatic, a bridgedbicyclic, 6-10 membered aryl, 5-10 membered heteroaryl having 1-4heteroatoms independently selected from nitrogen, oxygen, or sulfur, or4-7 membered heterocyclyl having 1-2 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur; and R⁷ is an optionally substitutedgroup selected from C₁₋₆ aliphatic, a bridged bicyclic, 6-10 memberedaryl, 5-10 membered heteroaryl having 1-4 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur, or 4-7 membered heterocyclylhaving 1-2 heteroatoms independently selected from nitrogen, oxygen, orsulfur; and R^(z) is

or R⁴ and R^(z) are taken together with their intervening atoms to forman optionally substituted, saturated or unsaturated 16-22 membered ringhaving 2-6 heteroatoms independently selected from nitrogen, oxygen, orsulfur; each occurrence of R^(y) is independently selected from halogen,—OR^(◯), —CN, —NO₂, —N(R^(◯))₂, or optionally substituted C₁₋₄aliphatic; R^(◯) is an optionally substituted group selected from C₁₋₆aliphatic; m is an integer from 0 to 4, inclusive; s is an integer from0 to 4, inclusive; t is an integer from 0 to 4, inclusive; wherein thesum of s and t is non-zero.
 30. The method of claim 29, wherein the stepof administering occurs once daily.
 31. The method according to claim29, wherein the disorder is hepatitis C.
 32. The method of claim 29,wherein the compound irreversibly inhibits HCV protease by covalentlymodifying a cysteine residue conserved at an equivalent position toCys159 of HCV protease subtype 1b.
 33. The method of claim 32, whereinthe HCV protease genotype or subtype is selected from the groupconsisting of 1a, 1b, 1c, 2a, 2b, 2c, 2i, 2k, 3a, 3b, 3k, 4a, 4d, 4f,5a, 6a, 6b, 6c, 6d, 6e, 6f, 6g, 6h, 6i, 6j, 6k, 6l, 6m, 6n, 6o, 6p, 6q,6t, and 7a.
 34. The method of claim 33, wherein the HCV proteasegenotype or subtype is 1a, 1b, 2a, or 3a.
 35. The method according toclaim 29, comprising the step of irreversibly inhibiting HCV protease bycovalently modifying Cys159 of HCV protease.
 36. The method according toclaim 29, comprising the step of irreversibly inhibiting HCV protease bycovalently modifying Cys16 of HCV protease. 37-47. (canceled)
 48. Themethod according to claim 25, wherein in the compound of formula I, R⁴and R^(z) are taken together with their intervening atoms to form anoptionally substituted, saturated or unsaturated 16-22 membered ringhaving 2-6 heteroatoms independently selected from nitrogen, oxygen, orsulfur.
 49. The method according to claim 48, wherein the compound is offormula I-e or I-f:

or a pharmaceutically acceptable salt thereof; wherein: p is an integerfrom 1 to 6, inclusive; and each occurrence of R^(m) is independentlyhalogen, —OR^(◯); —CN; —N(R^(◯))₂; or an optionally substituted groupselected from the group consisting of C₁₋₆ aliphatic and C₃₋₇cycloalkyl.
 50. The method according to claim 25, wherein in thecompound of formula I, R¹ and R^(1′) are taken together to form anoptionally substituted 3-7 membered carbocyclic ring.
 51. The methodaccording to claim 50, wherein in the compound of formula I, R¹ andR^(1′) are taken together to form:


52. The method according to claim 51, wherein in the compound of formulaI, R^(◯) is vinyl.
 53. The method according to claim 45, wherein in thecompound of formula I, R^(◯) is ethyl.
 54. The method according to claim25, wherein in the compound of formula I, R^(2a) is —NHSO₂R² and R² iscyclopropyl.
 55. The method according to claim 25, wherein in thecompound of formula I, R^(z) is


56. The method according to claim 25, wherein in the compound of formulaI, R⁴ is —NHC(O)OR⁶ and R⁶ is C₁₋₆ aliphatic.
 57. A method forinhibiting HCV protease, or a mutant thereof, in a patient comprisingthe step of administering to a patient in need thereof a compound offormula I:

or a pharmaceutically acceptable salt thereof, wherein: R¹ and R^(1′)are independently hydrogen or optionally substituted C₁₋₆ aliphatic, orR¹ and R^(1′) are taken together to form an optionally substituted 3-7membered carbocyclic ring; R^(2a) is —OH or —NHSO₂R²; R² is —N(R)₂ or anoptionally substituted group selected from C₃₋₇ cycloalkyl, a bridgedbicyclic, 6-10 membered aryl, 5-10 membered heteroaryl having 1-4heteroatoms independently selected from nitrogen, oxygen, or sulfur, or4-7 membered heterocyclyl having 1-2 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur; each R is independently hydrogen,optionally substituted C₁₋₆ aliphatic, or: two R on the same nitrogenatom are taken together with the nitrogen to form a 4-7 memberedheterocyclic ring having 1-2 heteroatoms independently selected fromnitrogen, oxygen, or sulfur; R³ is

R⁴ is H, —NHC(O)R⁵, —NHC(O)OR⁶,

or a natural or unnatural amino acid side-chain group; each R⁵ isindependently —N(R)₂ or an optionally substituted group selected fromC₁₋₆ aliphatic, a bridged bicyclic, 6-10 membered aryl, 5-10 memberedheteroaryl having 1-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur, or 4-7 membered heterocyclyl having 1-2 heteroatomsindependently selected from nitrogen, oxygen, or sulfur; R⁶ is anoptionally substituted group selected from C₁₋₆ aliphatic, a bridgedbicyclic, 6-10 membered aryl, 5-10 membered heteroaryl having 1-4heteroatoms independently selected from nitrogen, oxygen, or sulfur, or4-7 membered heterocyclyl having 1-2 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur; and R⁷ is an optionally substitutedgroup selected from C₁₋₆ aliphatic, a bridged bicyclic, 6-10 memberedaryl, 5-10 membered heteroaryl having 1-4 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur, or 4-7 membered heterocyclylhaving 1-2 heteroatoms independently selected from nitrogen, oxygen, orsulfur; and R^(z) is

and R⁴ and R^(z) are taken together with their intervening atoms to forman optionally substituted, saturated or unsaturated 16-22 membered ringhaving 2-6 heteroatoms independently selected from nitrogen, oxygen, orsulfur; each occurrence of R^(y) is independently selected from halogen,—OR^(◯), —CN, —NO₂, —N(R^(◯))₂, or optionally substituted C₁₋₄aliphatic; R^(◯) is an optionally substituted group selected from C₁₋₆aliphatic; m is an integer from 0 to 4, inclusive; s is an integer from0 to 4, inclusive; t is an integer from 0 to 4, inclusive; and whereinthe sum of s and t is non-zero.
 58. A method for inhibiting HCVprotease, or a mutant thereof, in a patient comprising the step ofadministering to a patient in need thereof a compound selected from thegroup consisting of:

or a pharmaceutically acceptable salt thereof.
 59. A method forinhibiting HCV protease, or a mutant thereof, activity in a biologicalsample comprising the step of contacting said biological sample with acompound of formula I:

or a pharmaceutically acceptable salt thereof, wherein: R¹ and R^(1′)are independently hydrogen or optionally substituted C₁₋₆ aliphatic, orR¹ and R^(1′) are taken together to form an optionally substituted 3-7membered carbocyclic ring; R^(2a) is —OH or —NHSO₂R²; R² is —N(R)₂ or anoptionally substituted group selected from C₃₋₇ cycloalkyl, a bridgedbicyclic, 6-10 membered aryl, 5-10 membered heteroaryl having 1-4heteroatoms independently selected from nitrogen, oxygen, or sulfur, or4-7 membered heterocyclyl having 1-2 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur; each R is independently hydrogen,optionally substituted C₁₋₆ aliphatic, or: two R on the same nitrogenatom are taken together with the nitrogen to form a 4-7 memberedheterocyclic ring having 1-2 heteroatoms independently selected fromnitrogen, oxygen, or sulfur; R³ is selected from the group consistingof:

R⁴ is H, —NHC(O)R⁵, —NHC(O)OR⁶,

or a natural or unnatural amino acid side-chain group; each R⁵ isindependently —N(R)₂ or an optionally substituted group selected fromC₁₋₆ aliphatic, a bridged bicyclic, 6-10 membered aryl, 5-10 memberedheteroaryl having 1-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur, or 4-7 membered heterocyclyl having 1-2 heteroatomsindependently selected from nitrogen, oxygen, or sulfur; R⁶ is anoptionally substituted group selected from C₁₋₆ aliphatic, a bridgedbicyclic, 6-10 membered aryl, 5-10 membered heteroaryl having 1-4heteroatoms independently selected from nitrogen, oxygen, or sulfur, or4-7 membered heterocyclyl having 1-2 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur; and R⁷ is an optionally substitutedgroup selected from C₁₋₆ aliphatic, a bridged bicyclic, 6-10 memberedaryl, 5-10 membered heteroaryl having 1-4 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur, or 4-7 membered heterocyclylhaving 1-2 heteroatoms independently selected from nitrogen, oxygen, orsulfur; and R^(z) is

or R⁴ and R^(z) are taken together with their intervening atoms to forman optionally substituted, saturated or unsaturated 16-22 membered ringhaving 2-6 heteroatoms independently selected from nitrogen, oxygen, orsulfur; each occurrence of R^(y) is independently selected from halogen,—OR^(◯), —CN, —NO₂, —N(R^(◯))₂, or optionally substituted C₁₋₄aliphatic; R^(◯) is an optionally substituted group selected from C₁₋₆aliphatic; m is an integer from 0 to 4, inclusive; s is an integer from0 to 4, inclusive; t is an integer from 0 to 4, inclusive; wherein thesum of s and t is non-zero.